Steroids In addition to fats, phospholipids, eicosanoids, and terpenoids, the lipid extracts of plants and animals also contain steroids , molecules that are derived from the triterpenoid lanosterol ( ) and whose structures are based on a tetracyclic ring system. The four rings are designated A, B, C, and D, beginning at the lower left. The three 6-membered rings (A, B, and C) adopt chair conformations but are prevented by their rigid geometry from undergoing the usual cyclohexane ring-flips ( ). Two cyclohexane rings can be joined in either a cis or a trans manner. With cis fusion to give cis -decalin, both groups at the ring-junction positions (the angular groups) are on the same side of the two rings. With trans fusion to give trans -decalin, the groups at the ring junctions are on opposite sides. As shown in , steroids can have either a cis or a trans fusion of the A and B rings, but the other ring fusions (B–C and C–D) are usually trans. Steroid conformations. The three 6-membered rings have chair conformations but are unable to undergo ring-flips. The A and B rings can be either cis-fused or trans-fused. Substituent groups on the steroid ring system can be either axial or equatorial. As with simple cyclohexanes, equatorial substitution is generally more favorable than axial substitution for steric reasons. The hydroxyl group of cholesterol, for example, has the more stable equatorial orientation. Problems Steroid Hormones In humans, most steroids function as hormones , chemical messengers that are secreted by endocrine glands and carried through the bloodstream to target tissues. There are two main classes of steroid hormones: the sex hormones, which control maturation, tissue growth, and reproduction, and the adrenocortical hormones, which regulate a variety of metabolic processes. Sex Hormones Testosterone and androsterone are the two most important male sex hormones, or androgens . Androgens are responsible for the development of male secondary sex characteristics during puberty and for promoting tissue and muscle growth. Both are synthesized in the testes from cholesterol. Androstenedione is another minor hormone that has received particular attention because of its use by prominent athletes. Estrone and estradiol are the two most important female sex hormones, or estrogens . Synthesized in the ovaries from testosterone, estrogenic hormones are responsible for the development of female secondary sex characteristics and for regulation of the menstrual cycle. Note that both have a benzene-like aromatic A ring. In addition, another kind of sex hormone called a progestin is essential in preparing the uterus for implantation of a fertilized ovum during pregnancy. Progesterone is the most important progestin. Adrenocortical Hormones Adrenocortical steroids are secreted by the adrenal glands, small organs located near the upper end of each kidney. There are two types of adrenocortical steroids, called mineralocorticoids and glucocorticoids . Mineralocorticoids, such as aldosterone, control tissue swelling by regulating cellular salt balance between Na + and K + . Glucocorticoids, such as hydrocortisone, are involved in the regulation of glucose metabolism and in the control of inflammation. Glucocorticoid ointments are widely used to bring down the swelling from exposure to poison oak or poison ivy. Synthetic Steroids In addition to the hundreds of steroids isolated from plants and animals, many more have been synthesized in pharmaceutical laboratories in the search for new drugs. Among the best-known synthetic steroids are oral contraceptives and anabolic agents. Most birth control pills are a mixture of two compounds, a synthetic estrogen, such as ethynylestradiol, and a synthetic progestin, such as norethindrone. Anabolic steroids , such as methandrostenolone (Dianabol), are synthetic androgens that mimic the tissue-building effects of natural testosterone.

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Biosynthesis of Steroids Steroids are heavily modified triterpenoids that are biosynthesized in living organisms from farnesyl diphosphate (C 15 ). A reductive dimerization first converts farnesyl diphosphate to the acyclic hydrocarbon squalene (C 30 ), which is converted into lanosterol ( ). Further rearrangements and degradations then take place to yield various steroids. The conversion of squalene to lanosterol is among the most intensively studied of biosynthetic transformations. Starting from an achiral, open-chain polyene, the entire process requires only two enzymes and results in the formation of six carbon–carbon bonds, four rings, and seven chirality centers. An overview of steroid biosynthesis from farnesyl diphosphate. Lanosterol biosynthesis begins with the selective epoxidation of squalene to give (3 S )-2,3-oxidosqualene, catalyzed by squalene epoxidase. Molecular O 2 provides the epoxide oxygen atom, and NADPH is required, along with a flavin coenzyme. The proposed mechanism involves reaction of FADH 2 with O 2 to produce a flavin hydroperoxide intermediate (ROOH), which transfers an oxygen to squalene in a pathway initiated by nucleophilic attack of the squalene double bond on the terminal hydroperoxide oxygen ( ). The flavin alcohol formed as a by-product loses H 2 O to give FAD, which is reduced back to FADH 2 by NADPH. As noted in , this biological epoxidation mechanism is closely analogous to the mechanism by which peroxyacids (RCO 3 H) react with alkenes to give epoxides in the laboratory. The second part of lanosterol biosynthesis is catalyzed by oxidosqualene-lanosterol cyclase and occurs as shown in . Squalene is folded by the enzyme into a conformation that aligns the various double bonds for a cascade of successive intramolecular electrophilic additions, followed by a series of hydride and methyl migrations. Except for the initial epoxide protonation/cyclization, the process is probably stepwise and appears to involve discrete carbocation intermediates that are stabilized by electrostatic interactions with electron-rich aromatic amino acids in the enzyme. Proposed mechanism of the oxidation of squalene by flavin hydroperoxide. Steps 1 , 2 of : Epoxide Opening and Initial Cyclizations Cyclization begins in step 1 with protonation of the epoxide ring by an aspartic acid residue in the enzyme. Nucleophilic opening of the protonated epoxide by the nearby 5,10 double bond (steroid numbering; ) then yields a tertiary carbocation at C10. Further addition of C10 to the 8,9 double bond in step 2 next gives a bicyclic tertiary cation at C8. Step 3 of : Third Cyclization The third cationic cyclization is somewhat unusual because it occurs with non-Markovnikov regiochemistry and gives a secondary cation at C13 rather than the alternative tertiary cation at C14. There is growing evidence, however, that the tertiary carbocation may in fact be formed initially and that the secondary cation arises by subsequent rearrangement. The secondary cation is probably stabilized in the enzyme pocket by the proximity of an electron-rich aromatic ring. MECHANISM Mechanism of the conversion of 2,3-oxidosqualene to lanosterol. Four cationic cyclizations are followed by four rearrangements and a final loss of H + from C9. The steroid numbering system is used for referring to specific positions in the intermediates ( ). Individual steps are explained in the text. (Continued) Step 4 of : Final Cyclization The fourth and last cyclization occurs in step 4 by addition of the cationic center at C13 to the 17,20 double bond, giving what is known as the protosteryl cation. The side-chain alkyl group at C17 has β (up) stereochemistry, although this stereochemistry is lost in step 5 and then reset in step 6. Steps 5 – 9 of : Carbocation Rearrangements Once the tetracyclic carbon skeleton of lanosterol has been formed, a series of carbocation rearrangements occur ( ). The first rearrangement, hydride migration from C17 to C20, occurs in step 5 and results in establishment of R stereochemistry at C20 in the side chain. In step 6, a second hydride migration occurs from C13 to C17 on the α (bottom) face of the ring and reestablishes the 17 β orientation of the side chain. Finally, two methyl migrations, the first from C14 to C13 on the top ( β ) face and the second from C8 to C14 on the bottom ( α ) face, place the positive charge at C8. A basic histidine residue in the enzyme then removes the neighboring β proton from C9 to give lanosterol. From lanosterol, the pathway for steroid biosynthesis continues on to yield cholesterol. Cholesterol then becomes a branch point, serving as the common precursor from which all other steroids are derived. Problems Saturated Fats, Cholesterol, and Heart Disease We hear a lot these days about the relationship between saturated fats, cholesterol, and heart disease. What are the facts? It’s well established that a diet rich in saturated animal fats often leads to an increase in blood serum cholesterol, particularly in sedentary, overweight individuals. Conversely, a diet lower in saturated fats and higher in polyunsaturated fats leads to a lower serum cholesterol level. Studies have shown that a serum cholesterol level greater than 240 mg/dL (a desirable value is < 200 mg/dL) is correlated with an increased incidence of coronary artery disease, in which cholesterol deposits build up on the inner walls of coronary arteries, blocking the flow of blood to the heart muscles. A better indication of a person’s risk of heart disease comes from a measurement of blood lipoprotein levels. Lipoproteins are complex molecules with both lipid and protein components that transport lipids through the body. They can be divided into three types according to density, as shown in . Very-low-density lipoproteins (VLDLs) act primarily as carriers of triglycerides from the intestines to peripheral tissues, whereas low-density lipoproteins (LDLs) and high-density lipoproteins (HDLs) act as carriers of cholesterol to and from the liver. It’s hard to resist, but a high intake of saturated animal fat doesn’t do much for your cholesterol level. (credit: modification of work “food-chicken-meat-outdoors” by www.pixel.la/Flickr, Public Domain) Evidence suggests that LDLs transport cholesterol as its fatty-acid ester to peripheral tissues, whereas HDLs remove cholesterol as its stearate ester from dying cells. If LDLs deliver more cholesterol than is needed, and if insufficient HDLs are present to remove it, the excess is deposited in arteries. Thus, a low level of low -density lipoproteins is good because it means that less cholesterol is being transported, and a high level of high -density lipoproteins is good because it means that more cholesterol is being removed. In addition, HDL contains an enzyme that has antioxidant properties, offering further protection against heart disease. As a rule of thumb, a person’s risk drops about 25% for each increase of 5 mg/dL in HDL concentration. Normal values are >40 mg/dL for men and <50 mg/dL for women, perhaps explaining why premenopausal women appear to be somewhat less susceptible than men to heart disease. Serum Lipoproteins Name Density (g/mL) % Lipid % Protein Optimal (mg/dL) Poor (mg/dL) VLDL 0.930–1.006 90 10 — — LDL 1.019–1.063 75 25 <100 >130 HDL 1.063–1.210 67 33 >60 <40 Not surprisingly, the most important factor in gaining high HDL levels is a generally healthful lifestyle. Obesity, smoking, and lack of exercise lead to low HDL levels, whereas regular exercise and a sensible diet lead to high HDL levels. Distance runners and other endurance athletes have HDL levels nearly 50% higher than the general population. Failing that—some of us, but not everyone, wants to run 30 miles or bike 80 miles per week—diet is also important. Diets high in cold-water fish, like salmon and whitefish, raise HDL and lower blood cholesterol because these fish contain almost entirely polyunsaturated fat, including a large percentage of omega-3 fatty acids. Animal fat from red meat and cooking fats should be minimized because saturated fats and monounsaturated trans fats raise blood cholesterol. anabolic steroid androgen eicosanoid estrogen fatty acid hormone lipid lipid bilayer lipoprotein micelle mineralocorticoid omega-3 fatty acid phospholipid polyunsaturated fatty acid progestin prostaglandin saponification steroid terpenoid triacylglycerol wax Summary Lipids are the naturally occurring materials isolated from plants and animals by extraction with a nonpolar organic solvent. Animal fats and vegetable oils are the most widely occurring lipids. Both are triacylglycerols —triesters of glycerol with long-chain fatty acids . Animal fats are usually saturated, whereas vegetable oils usually have unsaturated fatty acid residues. Phospholipids are important constituents of cell membranes and are of two kinds. Glycerophospholipids, such as phosphatidylcholine and phosphatidylethanolamine, are closely related to fats in that they have a glycerol backbone esterified to two fatty acids (one saturated and one unsaturated) and to one phosphate ester. Sphingomyelins have the amino alcohol sphingosine for their backbone. Eicosanoids and terpenoids are still other classes of lipids. Eicosanoids, of which prostaglandins are the most abundant kind, are derived biosynthetically from arachidonic acid, are found in all body tissues, and have a wide range of physiological activity. Terpenoids are often isolated from the essential oils of plants, have an immense diversity of structure, and are produced biosynthetically from the five-carbon precursor isopentenyl diphosphate (IPP). Isopentenyl diphosphate is itself biosynthesized from 3 equivalents of acetate in the mevalonate pathway. Steroids are plant and animal lipids with a characteristic tetracyclic carbon skeleton. Like the eicosanoids, steroids occur widely in body tissues and have a large variety of physiological activities. Steroids are closely related to terpenoids and arise biosynthetically from the triterpenoid lanosterol. Lanosterol, in turn, arises from cationic cyclization of the acyclic hydrocarbon squalene. Additional Problems Visualizing Chemistry Mechanism Problems Fats, Oils, and Related Lipids Terpenoids and Steroids General Problems

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Why This Chapter? If these Afghan hounds look similar, that’s because they’re identical—all cloned from somatic cells of the same donor . (credit: modification of work “Figure 1” in “Birth of clones of the world’s first cloned dog” by Kim, M.J., Oh, H.J., Kim, G.A. et al./ Sci Rep 7, 15235 (2017), CC BY 4.0) Nucleic acids are the last of the four major classes of biomolecules we’ll consider. So much has been written and spoken about DNA in the media that the basics of DNA replication and transcription are probably known to you. Thus, we’ll move fairly quickly through the fundamentals and then look more closely at the chemical details of DNA sequencing, synthesis, and metabolism. This field is moving rapidly, and there’s a lot you may not be familiar with. The nucleic acids, deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) , are the chemical carriers of a cell’s genetic information. Coded in a cell’s DNA is the information that determines the nature of the cell, controls its growth and division, and directs biosynthesis of the enzymes and other proteins required for cellular functions. In addition to nucleic acids themselves, nucleic acid derivatives such as ATP are involved as phosphorylating agents in many biochemical pathways, and several important coenzymes, including NAD + , FAD, and coenzyme A, have nucleic acid components. See for their structures.

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Nucleotides and Nucleic Acids Just as proteins are biopolymers made of amino acids, the nucleic acids are biopolymers made of nucleotides , joined together to form a long chain. Each nucleotide is composed of a nucleoside bonded to a phosphate group, and each nucleoside is composed of an aldopentose sugar linked through its anomeric carbon to the nitrogen atom of a heterocyclic purine or pyrimidine base. The sugar component in RNA is ribose, and the sugar in DNA is 2′-deoxyribose. (In naming and numbering nucleotides, numbers with a prime superscript refer to positions on the sugar and numbers without a prime superscript refer to positions on the heterocyclic base. Thus, the prefix 2′-deoxy indicates that oxygen is missing from C2′ of ribose.) DNA contains four different amine bases: two substituted purines ( A , adenine, and G , guanine) and two substituted pyrimidines ( C , cytosine, and T , thymine). Adenine, guanine, and cytosine also occur in RNA, but thymine is replaced in RNA by a closely related pyrimidine base called U , uracil. The structures of the four deoxyribonucleotides and the four ribonucleotides are shown in . Although similar chemically, DNA and RNA differ dramatically in size. Molecules of DNA are enormous, containing as many as 245 million nucleotides and having molecular weights as high as 75 billion. Molecules of RNA, by contrast, are much smaller, containing as few as 21 nucleotides and having molecular weights as low as 7000. Structures of the four deoxyribonucleotides and the four ribonucleotides. Nucleotides are linked together in DNA and RNA by phosphodiester bonds [RO–(PO 2 – )–OR′] between phosphate, the 5′-hydroxyl group on one nucleoside, and the 3′-hydroxyl group on another nucleoside. One end of the nucleic acid polymer has a free hydroxyl at C3′ (the 3′ end ), and the other end has a phosphate at C5′ (the 5′ end ). The sequence of nucleotides in a chain is described by starting at the 5′ end and identifying the bases in order of occurrence, using the abbreviations G, C, A, T (or U in RNA). Thus, a typical DNA sequence might be written as TAGGCT. Problems

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Base Pairing in DNA Samples of DNA isolated from different tissues of the same species have the same proportions of heterocyclic bases, but samples from different species often have greatly differing proportions of bases. Human DNA, for example, contains about 30% each of adenine and thymine and about 20% each of guanine and cytosine. The bacterium Clostridium perfringens, however, contains about 37% each of adenine and thymine and only 13% each of guanine and cytosine. Note that in both examples the bases occur in pairs. Adenine and thymine are present in equal amounts, as are cytosine and guanine. Why? In 1953, Rosalind Franklin, Maurice Wilkins, James Watson, and Francis Crick published scientific reports describing the secondary structure of DNA. According to their model, DNA under physiological conditions consists of two polynucleotide strands, running in opposite directions and coiled around each other in a double helix like the handrails on a spiral staircase. The two strands are complementary rather than identical and are held together by hydrogen bonds between specific pairs of bases, A with T and C with G. That is, whenever an A base occurs in one strand, a T base occurs opposite it in the other strand; when a C base occurs in one, a G occurs in the other ( ). This complementary base-pairing thus explains why A and T are always found in equal amounts, as are G and C. Hydrogen-bonding between base pairs in the DNA double helix. Electrostatic potential maps show that the faces of the bases are relatively neutral , while the edges have positive and negative regions. Pairing G with C and A with T brings together oppositely charged regions. A full turn of a DNA double helix is shown in . The helix is 20 Å wide, there are 10 base pairs per turn, and each turn is 34 Å in length. Notice in that the two strands of the double helix coil in such a way that two kinds of “grooves” result, a major groove 12 Å wide and a minor groove 6 Å wide. The major groove is slightly deeper than the minor groove, and both are lined by flat heterocyclic bases. As a result, a variety of other polycyclic aromatic molecules are able to slip sideways, or intercalate, between the stacked bases. Many cancer-causing and cancer-preventing agents function by interacting with DNA in this way. A turn of the DNA double helix in both space-filling and wire-frame formats. The sugar–phosphate backbone runs along the outside of the helix, and the amine bases hydrogen bond to one another on the inside. Both major and minor grooves are visible. An organism’s genetic information is stored as a sequence of deoxyribonucleotides strung together in the DNA chain. For the information to be preserved and passed on to future generations, a mechanism must exist for copying DNA. For the information to be used, a mechanism must exist for decoding the DNA message and implementing the instructions it contains. What Crick called the “central dogma of molecular genetics” says that the function of DNA is to store information and pass it on to RNA. The function of RNA, in turn, is to read, decode, and use the information received from DNA to make proteins. This view is greatly oversimplified but is nevertheless a good place to start. Three fundamental processes take place. Replication is the process by which identical copies of DNA are made so that information can be preserved and handed down to offspring. Transcription is the process by which genetic messages are read and carried out of the cell nucleus to ribosomes, where protein synthesis occurs. Translation is the process by which the genetic messages are decoded and used to synthesize proteins. Predicting the Complementary Base Sequence in Double-Stranded DNA What sequence of bases on one strand of DNA is complementary to the sequence TATGCAT on another strand? Strategy Remember that A and G form complementary pairs with T and C, respectively, and then go through the sequence replacing A by T, G by C, T by A, and C by G. Remember also that the 5′ end is on the left and the 3′ end is on the right in the original strand. Solution Original: (5′) TATGCAT (3′) Complement: (3′) ATACGTA (5′) or (5′) ATGCATA (3′) Problems

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Replication of DNA DNA replication is an enzyme-catalyzed process that begins with a partial unwinding of the double helix at various points along the chain, brought about by enzymes called helicases. Hydrogen bonds are broken, the two strands separate to form a “bubble,” and bases are exposed. New nucleotides then line up on each strand in a complementary manner, A to T and G to C, and two new strands begin to grow from the ends of the bubble, called the replication forks . Each new strand is complementary to its old template strand, so two identical DNA double helices are produced ( ). Because each of the new DNA molecules contains one old strand and one new strand, the process is described as semiconservative replication. A representation of semiconservative DNA replication. The original double-stranded DNA partially unwinds, bases are exposed, nucleotides line up on each strand in a complementary manner, and two new strands begin to grow. Both strands are synthesized in the same 5′ → 3′ direction, one continuously and one in fragments. Addition of nucleotides to the growing chain takes place in the 5′ → 3′ direction and is catalyzed by the DNA polymerase enzyme. The key step is the addition of a nucleoside 5′-triphosphate to the free 3′-hydroxyl group of the growing chain with the loss of a diphosphate leaving group. Because both new DNA strands are synthesized in the 5′ → 3′ direction, they can’t be made in exactly the same way. One new strand must have its 3′ end nearer a replication fork, while the other new strand has its 5′ end nearer the replication fork. What happens is that the complement of the original 5′ → 3′ strand is synthesized continuously in a single piece to give a newly synthesized copy called the leading strand , while the complement of the original 3′ → 5′ strand is synthesized discontinuously in small pieces called Okazaki fragments (named for Tsuneko Okazaki, in recognition of her discovery of them) that are subsequently linked by DNA ligases to form the lagging strand . The magnitude of the replication process is staggering. The nucleus of every human cell contains 2 copies of 22 chromosomes plus an additional 2 sex chromosomes, for a total of 46. Each chromosome consists of one very large DNA molecule, and the sum of the DNA in each of the two sets of chromosomes is estimated to be 3.0 billion base pairs, or 6.0 billion nucleotides. Despite the size of these enormous molecules, their base sequence is faithfully copied during replication. The entire copying process takes only a few hours and, after proofreading and repair, an error gets through only about once per 10 to 100 billion bases. In fact, only about 60 of these random mutations are passed on from parent to child per human generation.

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Transcription of DNA As noted previously, RNA is structurally similar to DNA but contains ribose rather than deoxyribose and uracil rather than thymine. RNA has three major types, each of which serves a specific purpose. In addition, there are a number of small RNAs that appear to control a wide variety of important cellular functions. All RNA molecules are much smaller than DNA, and all remain single-stranded rather than double-stranded. Messenger RNA (mRNA) carries genetic messages from DNA to ribosomes, small granular particles in the cytoplasm of a cell where protein synthesis takes place. Ribosomal RNA (rRNA) complexed with protein provides the physical makeup of the ribosomes. Transfer RNA (tRNA) transports amino acids to the ribosomes, where they are joined together to make proteins. Small RNAs , also called functional RNAs , have a variety of functions within the cell, including silencing transcription and catalyzing chemical modifications of other RNA molecules. The genetic information in DNA is contained in segments called genes , each of which consists of a specific nucleotide sequence that encodes a specific protein. The conversion of that information from DNA into proteins begins in the nucleus of cells with the synthesis of mRNA by transcription of DNA. In bacteria, the process begins when RNA polymerase recognizes and binds to a promoter sequence on DNA, typically consisting of around 40 base pairs located upstream (5′) of the transcription start site. Within the promoter are two hexameric consensus sequences , one located 10 base pairs upstream of the start and the second located 35 base pairs upstream. Following formation of the polymerase–promoter complex, several turns of the DNA double helix untwist, forming a bubble and exposing 14 or so base pairs of the two strands. Appropriate ribonucleotides then line up by hydrogen-bonding to their complementary bases on DNA, bond formation occurs in the 5′ → 3′ direction, the RNA polymerase moves along the DNA chain, and the growing RNA molecule unwinds from DNA ( ). At any one time, about 12 base pairs of the growing RNA remain hydrogen-bonded to the DNA template. Biosynthesis of RNA using a DNA segment as a template. Unlike what happens in DNA replication, where both strands are copied, only one of the two DNA strands is transcribed into mRNA. The DNA strand that contains the gene is often called the sense strand , or coding strand, and the DNA strand that gets transcribed to give RNA is called the antisense strand , or noncoding strand. Because the sense strand and the antisense strand in DNA are complementary, and because the DNA antisense strand and the newly formed RNA strand are also complementary, the RNA molecule produced during transcription is a copy of the DNA sense strand. That is, the complement of the complement is the same as the original. The only difference is that the RNA molecule has a U everywhere that the DNA sense strand has a T. Another part of the picture in vertebrates and flowering plants is that genes are often not continuous segments of the DNA chain. Instead, a gene will begin in one small section of DNA called an exon, then be interrupted by a noncoding section called an intron, and then take up again farther down the chain in another exon. The final mRNA molecule results only after the noncoded sections are cut out of the transcribed mRNA and the remaining pieces are joined together by spliceosome enzymes. The gene for triose phosphate isomerase in maize, for instance, contains eight noncoding introns accounting for approximately 70% of the DNA base pairs and nine coding exons accounting for only 30% of the base pairs. Problems

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Translation of RNA: Protein Biosynthesis The primary cellular function of mRNA is to direct the biosynthesis of the thousands of diverse peptides and proteins required by an organism—as many as 150,000 in a human. The mechanics of protein biosynthesis take place on ribosomes, small granular particles in the cytoplasm of a cell that consist of about 60% ribosomal RNA and 40% protein. The specific ribonucleotide sequence in mRNA forms a message that determines the order in which amino acid residues are to be joined. Each “word,” or codon , along the mRNA chain consists of a sequence of three ribonucleotides that is specific for a given amino acid. For example, the series UUC on mRNA is a codon directing incorporation of the amino acid phenylalanine into the growing protein. Of the 4 3 = 64 possible triplets of the four bases in RNA, 61 code for specific amino acids and 3 code for chain termination. shows the meaning of each codon. Codon Assignments of Base Triplets Third base (3′ end) First base (5′ end) Second base U C A G U U Phe Phe Leu Leu C Ser Ser Ser Ser A Tyr Tyr Stop Stop G Cys Cys Stop Trp C U Leu Leu Leu Leu C Pro Pro Pro Pro A His His Gln Gln G Arg Arg Arg Arg A U Ile Ile Ile Met C Thr Thr Thr Thr A Asn Asn Lys Lys G Ser Ser Arg Arg G U Val Val Val Val C Ala Ala Ala Ala A Asp Asp Glu Glu G Gly Gly Gly Gly The message embedded in mRNA is read by transfer RNA (tRNA) in a process called translation . There are 61 different tRNAs, one for each of the 61 codons that specify an amino acid. A typical tRNA is single-stranded and has roughly the shape of a cloverleaf, as shown in . It consists of about 70 to 100 ribonucleotides and is bonded to a specific amino acid by an ester linkage through the 3′ hydroxyl on ribose at the 3′ end of the tRNA. Each tRNA also contains on its middle leaf a segment called an anticodon , a sequence of three ribonucleotides complementary to the codon sequence. For example, the codon sequence UUC present on mRNA is read by a phenylalanine-bearing tRNA having the complementary anticodon base sequence GAA. [Remember that nucleotide sequences are written in the 5′ → 3′ direction, so the sequence in an anticodon must be reversed. That is, the complement to (5′)-UUC-(3′) is (3′)-AAG-(5′), which is written as (5′)-GAA-(3′).] Structure of a tRNA molecule. The tRNA is a roughly cloverleaf-shaped molecule containing an anticodon triplet on one “leaf” and an amino acid unit attached covalently at its 3′ end. The example shown is a yeast tRNA that codes for phenylalanine. The nucleotides not specifically identified are chemically modified analogs of the four common nucleotides. As each successive codon on mRNA is read, different tRNAs bring the correct amino acids into position for enzyme-mediated transfer to the growing peptide. When synthesis of the proper protein is completed, a “stop” codon signals the end, and the protein is released from the ribosome. This process is illustrated in . A representation of protein biosynthesis. The codon base sequences on mRNA are read by tRNAs containing complementary anticodon base sequences. Transfer RNAs assemble the proper amino acids into position for incorporation into the growing peptide. Predicting the Amino Acid Sequence Transcribed from DNA What amino acid sequence is coded by the following segment of a DNA coding strand (sense strand)? (5′) CTA-ACT-AGC-GGG-TCG-CCG (3′) Strategy The mRNA produced during translation is a copy of the DNA coding strand, with each T replaced by U. Thus, the mRNA has the sequence (5′) CUA-ACU-AGC-GGG-UCG-CCG (3′) Each set of three bases forms a codon, whose meaning can be found in . Solution Leu-Thr-Ser-Gly-Ser-Pro. Problems

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DNA Sequencing A scientific revolution is now under way in molecular biology, as scientists learn how to manipulate and harness the genetic machinery of organisms. None of the extraordinary advances of the past several decades would have been possible, however, were it not for the discovery in 1977 of methods for sequencing immense DNA chains. The first step in DNA sequencing is to cleave the enormous chain at known points to produce smaller, more manageable pieces, a task accomplished by the use of restriction endonucleases , or restriction enzymes. Each different restriction enzyme, of which more than 4000 are known and approximately 600 are commercially available, cleaves a DNA molecule at a point in the chain where a specific base sequence occurs. For example, the restriction enzyme Alu I cleaves between G and C in the four-base sequence AG-CT. Note that the sequence is a palindrome, meaning that the sequence (5′)-AGCT-(3′) is the same as its complement (3′)-TCGA-(5′) when both are read in the same 5′ → 3′ direction. The same is true for other restriction endonucleases. If the original DNA molecule is cut with another restriction enzyme having a different specificity for cleavage, still other segments are produced whose sequences partially overlap those produced by the first enzyme. Sequencing all the segments, followed by identification of the overlapping regions, allows for complete DNA sequencing. A dozen or so different methods of DNA sequencing are now available, and many others are under development. The Sanger dideoxy method is among the most frequently used and was the method responsible for first sequencing the entire human genome of 3.0 billion base pairs. In commercial sequencing instruments, the dideoxy method begins with a mixture of the following: The restriction fragment to be sequenced A small piece of DNA called a primer, whose sequence is complementary to that on the 3′ end of the restriction fragment The four 2′-deoxyribonucleoside triphosphates (dNTPs) Very small amounts of the four 2′,3′-dideoxyribonucleoside triphosphates (ddNTPs), each of which is labeled with a fluorescent dye of a different color (A 2′,3′- dideoxy ribonucleoside triphosphate is one in which both 2′ and 3′ –OH groups are missing from ribose.) DNA polymerase is added to the mixture, and a strand of DNA complementary to the restriction fragment begins to grow from the end of the primer. Most of the time, only normal deoxyribonucleotides are incorporated into the growing chain because of their much higher concentration in the mixture, but every so often, a dideoxyribonucleotide is incorporated. When that happens, DNA synthesis stops because the chain end no longer has a 3′-hydroxyl group for adding further nucleotides. When reaction is complete, the product consists of a mixture of DNA fragments of all possible lengths, each terminated by one of the four dye-labeled dideoxyribonucleotides. This product mixture is then separated according to the size of the pieces by gel electrophoresis ( ), and the identity of the terminal dideoxyribonucleotide in each piece—and thus the sequence of the restriction fragment—is determined by noting the color with which the attached dye fluoresces. shows a typical result. The sequence of a restriction fragment determined by the Sanger dideoxy method can be read simply by noting the colors of the dye attached to each of the various terminal nucleotides. So efficient is the automated dideoxy method that sequences up to 1100 nucleotides in length, with a throughput of up to 19,000 bases per hour, can be sequenced with 98% accuracy. After a decade of work and a cost of about $500 million, preliminary sequence information for the entire human genome of 3.0 billion base pairs was announced early in 2001 and complete information was released in 2003. More recently, the genome sequencing of individuals, including that of James Watson, one of the discoverers of the double helix, has been accomplished. The sequencing price per genome is dropping rapidly and is currently less than $1,000, meaning that the routine sequencing of individuals is within reach. Remarkably, our genome appears to contain only about 21,000 genes, less than one-fourth the previously predicted number and only about twice the number found in the common roundworm. It’s also interesting to note that the number of genes in a human (21,000) is much smaller than the number of kinds of proteins (perhaps 500,000). This discrepancy arises because most proteins are modified in various ways after translation (posttranslational modifications), so a single gene can ultimately give many different proteins.

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DNA Synthesis The ongoing revolution in molecular biology has brought with it an increased demand for the efficient chemical synthesis of short DNA segments, called oligonucleotides, or simply oligos. The problems of DNA synthesis are similar to those of peptide synthesis ( ) but are more difficult because of the complexity of the nucleotide monomers. Each nucleotide has multiple reactive sites that must be selectively protected and deprotected at specific times, and coupling of the four nucleotides must be carried out in the proper sequence. Automated DNA synthesizers are available, however, that allow the fast and reliable synthesis of DNA segments up to 200 nucleotides in length. DNA synthesizers operate on a principle similar to that of the Merrifield solid-phase peptide synthesizer ( ). In essence, a protected nucleotide is covalently bonded to a solid support, and one nucleotide at a time is added to the growing chain by the use of a coupling reagent. After the final nucleotide has been added, all the protecting groups are removed and the synthetic DNA is cleaved from the solid support. Five steps are needed: STEP 1 The first step in DNA synthesis is to attach a protected deoxynucleoside to a silica (SiO 2 ) support by an ester linkage to the 3′ –OH group of the deoxynucleoside. Both the 5′ –OH group on the sugar and free –NH 2 groups on the heterocyclic bases must be protected. Adenine and cytosine bases are protected by benzoyl groups, guanine is protected by an isobutyryl group, and thymine requires no protection. The deoxyribose 5′ –OH is protected as its p -dimethoxytrityl (DMT) ether. STEP 2 The second step is removal of the DMT protecting group by treatment with dichloroacetic acid in CH 2 Cl 2 . The reaction occurs by an S N 1 mechanism and proceeds rapidly because of the stability of the tertiary, benzylic dimethoxytrityl cation. STEP 3 The third step is the coupling of the polymer-bonded deoxynucleoside with a protected deoxynucleoside containing a phosphoramidite group [R 2 NP(OR) 2 ] at its 3′ position. The coupling reaction takes place in the polar aprotic solvent acetonitrile, requires catalysis by the heterocyclic amine tetrazole, and yields a phosphite, P(OR) 3 , as product. Note that one of the phosphorus oxygen atoms is protected by a β -cyanoethyl group, – OCH 2 CH 2 C≡N . The coupling step takes place with better than 99% yield. STEP 4 With the coupling accomplished, the phosphite product is oxidized to a phosphate by treatment with iodine in aqueous tetrahydrofuran in the presence of 2,6-dimethylpyridine. The cycle of ( 1 ) deprotection, ( 2 ) coupling, and ( 3 ) oxidation is then repeated until an oligonucleotide chain of the desired sequence has been constructed. STEP 5 The final step is removal of all protecting groups and cleavage of the ester bond holding the DNA to the silica. All these reactions are done at the same time by treatment with aqueous NH 3 . Purification by electrophoresis then yields the synthetic DNA. Problems

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The Polymerase Chain Reaction It often happens that only a tiny amount of DNA can be obtained directly, as might occur at a crime scene, so methods for obtaining larger amounts are sometimes needed to carry out sequencing and characterization. The invention of the polymerase chain reaction (PCR) by Kary Mullis in 1986 has been described as being to genes what Gutenberg’s invention of the printing press was to the written word. Just as the printing press produces multiple copies of a book, PCR produces multiple copies of a given DNA sequence. Starting from less than 1 picogram (pg) of DNA with a chain length of 10,000 nucleotides (1 pg = 10 –12 g; about 10 5 molecules), PCR makes it possible to obtain several micrograms (1 μ g = 10 –6 g; about 10 11 molecules) in just a few hours. The key to the polymerase chain reaction is Taq DNA polymerase, a heat-stable enzyme isolated from the thermophilic bacterium Thermus aquaticus found in a hot spring in Yellowstone National Park. Taq polymerase is able to take a single strand of DNA having a short, primer segment of complementary chain at one end and then finish constructing the entire complementary strand. The overall process takes three steps, as shown in . More recently, improved heat-stable DNA polymerases have become available, including Vent polymerase and Pfu polymerase, both isolated from bacteria growing near geothermal vents in the ocean floor. The error rate of both enzymes is substantially less than that of Taq . The polymerase chain reaction. Details are explained in the text. STEP 1 The double-stranded DNA to be amplified is heated in the presence of Taq polymerase, Mg 2+ ion, the four deoxynucleotide triphosphate monomers (dNTPs), and a large excess of two short oligonucleotide primers of about 20 bases each. Each primer is complementary to the sequence at the end of one of the target DNA segments. At a temperature of 95 °C, double-stranded DNA denatures, spontaneously breaking apart into two single strands. STEP 2 The temperature is lowered to between 37 and 50 °C, allowing the primers, because of their relatively high concentration, to anneal by hydrogen-bonding to their complementary sequence at the end of each target strand. STEP 3 The temperature is then raised to 72 °C, and Taq polymerase catalyzes the addition of further nucleotides to the two primed DNA strands. When replication of each strand is complete, two copies of the original DNA now exist. Repeating the denature–anneal–synthesize cycle a second time yields four DNA copies, repeating a third time yields eight copies, and so on, in an exponential series. PCR has been automated, and 30 or so cycles can be carried out in an hour, resulting in a theoretical amplification factor of 2 30 (∼10 9 ). In practice, however, the efficiency of each cycle is less than 100%, and an experimental amplification of about 10 6 to 10 8 is routinely achieved for 30 cycles. DNA Fingerprinting The invention of DNA sequencing has affected society in many ways, few more dramatic than those stemming from the development of DNA fingerprinting. DNA fingerprinting arose from the discovery in 1984 that human genes contain short, repeating sequences of noncoding DNA, called short tandem repeat (STR) loci. Furthermore, the STR loci are slightly different for everyone except identical twins. By sequencing these loci, a pattern unique to each person can be obtained. Perhaps the most common and well-publicized use of DNA fingerprinting is that carried out by crime laboratories to link suspects to biological evidence—blood, hair follicles, skin, or semen—found at a crime scene. Many thousands of court cases have been decided based on DNA evidence. For use in criminal cases, forensic laboratories in the United States have agreed on 13 core STR loci that are most accurate for the identification of an individual. Based on these 13 loci, a Combined DNA Index System (CODIS) has been established to serve as a registry of convicted offenders. When a DNA sample is obtained from a crime scene, the sample is subjected to cleavage with restriction endonucleases to cut out fragments containing the STR loci, the fragments are amplified using the polymerase chain reaction, and the sequences of the fragments are determined. If the profile of sequences from a known individual and the profile from DNA obtained at a crime scene match, the probability is approximately 82 billion to 1 that the DNA is from the same individual. In paternity cases, where the DNA of male parent and offspring are related but not fully identical, the identity of the male parent can be established with a probability of around 100,000 to 1. Even after several generations, paternity can still be inferred from DNA analysis of the Y chromosome of direct male-line descendants. The most well-known such case is that of Thomas Jefferson, one of the nation's founders, who likely impregnated enslaved person Sally Hemings. Although Jefferson himself has no male-line descendants, DNA analysis of the male-line descendants of Jefferson’s paternal uncle contained the same Y chromosome as a male-line descendant of Eston Hemings, the youngest son of Sally Hemings. Thus, a mixing of the two genomes is clear, although the male individual responsible for that mixing can’t be identified with 100% certainty. Among its many other applications, DNA fingerprinting is widely used for the diagnosis of genetic disorders, both prenatally and in newborns. Cystic fibrosis, hemophilia, Huntington’s disease, Tay–Sachs disease, sickle cell anemia, and thalassemia are among the many diseases that can be detected, enabling early treatment of an affected child. Furthermore, by studying the DNA fingerprints of relatives with a history of a particular disorder, it’s possible to identify DNA patterns associated with the disease and perhaps obtain clues for an eventual cure. In addition, the U.S. Department of Defense requires blood and saliva samples from all military personnel. The samples are stored, and DNA is extracted if the need for identification of a casualty arises. anticodon antisense strand codon deoxyribonucleic acid (DNA) double helix 3′ end 5′ end messenger RNA (mRNA) nucleoside nucleotides polymerase chain reaction (PCR) replication ribonucleic acid (RNA) ribosomal RNA (rRNA) Sanger dideoxy method sense strand small RNAs transcription transfer RNA (tRNA) translation Summary DNA (deoxyribonucleic acid) and RNA (ribonucleic acid) are biological polymers that act as chemical carriers of an organism’s genetic information. Enzyme-catalyzed hydrolysis of nucleic acids yields nucleotides , the monomer units from which RNA and DNA are constructed. Further enzyme-catalyzed hydrolysis of the nucleotides yields nucleosides plus phosphate. Nucleosides, in turn, consist of a purine or pyrimidine base linked to the C1 of an aldopentose sugar—ribose in RNA and 2-deoxyribose in DNA. The nucleotides are joined by phosphate links between the 5′ phosphate of one nucleotide and the 3′ hydroxyl on the sugar of another nucleotide. Molecules of DNA consist of two complementary polynucleotide strands held together by hydrogen bonds between heterocyclic bases on the different strands and coiled into a double helix . Adenine and thymine form hydrogen bonds to each other, as do cytosine and guanine. Three processes take place in deciphering the genetic information of DNA: Replication of DNA is the process by which identical DNA copies are made. The DNA double helix unwinds, complementary deoxyribonucleotides line up in order, and two new DNA molecules are produced. Transcription is the process by which RNA is produced to carry genetic information from the nucleus to the ribosomes. A short segment of the DNA double helix unwinds, and complementary ribonucleotides line up to produce messenger RNA (mRNA) . Translation is the process by which mRNA directs protein synthesis. Each mRNA is divided into codons , ribonucleotide triplets that are recognized by small amino acid–carrying molecules of transfer RNA (tRNA) , which deliver the appropriate amino acids needed for protein synthesis. Sequencing of DNA is carried out by the Sanger dideoxy method , and small DNA segments can be synthesized in the laboratory by automated instruments. Small amounts of DNA can be amplified by factors of 10 6 using the polymerase chain reaction (PCR) . Additional Problems Visualizing Chemistry Mechanism Problems General Problems

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Why This Chapter? Acyl CoA dehydrogenase is an enzyme that catalyzes the introduction of a C═C double bond into fatty acids during their metabolism . (credit: modification of image from the RCSB PDF (rcsb.org) of PDB ID 2WBI. Muniz, J.R.C., Guo, K., Savitsky, P., Roos, A., Yue, W., Pilka, E., Vondelft, F., Edwards, A.M., Bountra, C., Arrowsmith, C.H., Weigelt, J., Oppermann, U. Crystal structure of human Acyl-CoA dehydrogenase 11) In this chapter, we’ll look at some of the pathways by which organisms carry out their chemistry, focusing primarily on how they metabolize fats and carbohydrates. The treatment will be far from complete, but it should give you an idea of the kinds of processes that occur. Anyone who wants to understand or contribute to the revolution now taking place in the biological sciences must first understand life processes at the molecular level. This understanding, in turn, must be based on a detailed knowledge of the chemical reactions and pathways used by living organisms. Just knowing what occurs is not enough; it’s also necessary to understand how and why organisms use the chemistry they do. Biochemical reactions are not mysterious. Even though the biological reactions that take place in living organisms often appear complicated, they follow the same rules of reactivity as laboratory reactions and they operate by the same mechanisms. Some of the molecules we’ll be encountering are substantially larger and more complex than those we’ve been dealing with thus far. But don’t be intimidated; keep your focus on the parts of the molecules where changes occur, and ignore the parts where nothing changes. The reactions themselves are exactly the same additions, eliminations, substitutions, carbonyl condensations, and so forth, that we’ve been dealing with all along. By the end of this chapter, it should be clear that the chemistry of living organisms is organic chemistry.

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An Overview of Metabolism and Biochemical Energy The many reactions that occur in the cells of living organisms are collectively called metabolism . The pathways that break down larger molecules into smaller ones are called catabolism , and the pathways that synthesize larger biomolecules from smaller ones are known as anabolism . Catabolic reaction pathways are usually exergonic and release energy, while anabolic pathways are often endergonic and absorb energy. Catabolism can be divided into the four stages shown in . An overview of catabolic pathways for the degradation of food and the production of biochemical energy. The ultimate products of food catabolism are CO 2 and H 2 O, with the energy released in the citric acid cycle used to drive the endergonic synthesis of adenosine triphosphate (ATP) from adenosine diphosphate (ADP) plus hydrogen phosphate ion, HOPO 3 2– . In the first catabolic stage, commonly called digestion , food is broken down in the mouth, stomach, and small intestine by hydrolysis of ester, acetal (glycoside), and amide (peptide) bonds to yield fatty acids, simple sugars, and amino acids. These smaller molecules are then absorbed and further degraded in the second stage of catabolism to yield acetyl groups attached by a thioester bond to the large carrier molecule, coenzyme A. The resultant compound, acetyl coenzyme A (acetyl CoA), is a key substance in the metabolism of food molecules and in many other biological pathways. As noted in , the acetyl group in acetyl CoA is linked to the sulfur atom of phosphopantetheine, which is itself linked to adenosine 3',5'-bisphosphate. Acetyl groups are oxidized inside cellular mitochondria in the third stage of catabolism, the citric acid cycle, to yield CO 2 . (We’ll see the details of the process in Section 29.7 .) Like most oxidations, this stage releases a large amount of energy, which is used in the fourth stage, the electron-transport chain, to accomplish the endergonic phosphorylation of adenosine diphosphate (ADP) with hydrogen phosphate ion (HOPO 3 2– , abbreviated P i ) to give adenosine triphosphate (ATP). As the final result of food catabolism, ATP has been called the “energy currency” of the cell. Catabolic reactions “buy” ATP with the energy they release to synthesize it from ADP and hydrogen phosphate ion. Anabolic reactions then spend the ATP by transferring a phosphate group to another molecule, thereby regenerating ADP. Energy production and use in living organisms thus revolves around the ATP ⇄ ADP interconversion. ADP and ATP are both phosphoric acid anhydrides, which contain linkages analogous to the linkage in carboxylic acid anhydrides. Just as carboxylic acid anhydrides react with alcohols by breaking a C–O bond and forming a carboxylic ester, ROCOR' ( ), phosphoric acid anhydrides react with alcohols by breaking a P–O bond and forming a phosphate ester, ROPO 3 2– . The reaction is, in effect, a nucleophilic acyl substitution at phosphorus. Note that phosphorylation reactions with ATP generally require the presence of a divalent metal cation in the enzyme, usually Mg 2+ , to form a Lewis acid–base complex with the phosphate oxygen atoms and to neutralize negative charge. How does the body use ATP? Recall from that the free-energy change Δ G must be negative and energy must be released for a reaction to be energetically favorable and occur spontaneously. If Δ G is positive, the reaction is energetically unfavorable and the process can’t occur spontaneously. For an energetically unfavorable reaction to occur, it must be “coupled” to an energetically favorable reaction so that the overall free-energy change for the two reactions together is favorable. To understand what it means for reactions to be coupled, imagine that reaction 1 does not occur to any reasonable extent because it has a small equilibrium constant and is energetically unfavorable; that is, the reaction has Δ G > 0. where A and B are the biochemically “important” substances while m and n are enzyme cofactors, H 2 O, or other small molecules. Imagine also that product n can react with substance o to yield p and q in a second, highly favorable reaction that has a large equilibrium constant and Δ G << 0. Taking the two reactions together, they share, or are coupled through, the common intermediate n , which is a product in the first reaction and a reactant in the second. When even a tiny amount of n is formed in reaction 1, it undergoes essentially complete conversion in reaction 2, thereby removing it from the first equilibrium and forcing reaction 1 to continually replenish n until reactant A is gone. That is, the two reactions added together have a favorable Δ G < 0, and we say that the favorable reaction 2 “drives” the unfavorable reaction 1. Because the two reactions are coupled through n , the transformation of A to B becomes favorable. For an example of two reactions that are coupled, look at the phosphorylation reaction of glucose to yield glucose 6-phosphate plus water, an important step in the breakdown of dietary carbohydrates. The reaction of glucose with HOPO 3 2– does not occur spontaneously because it is energetically unfavorable, with Δ G °' = 13.8 kJ/mol. (The standard free-energy change for a biological reaction is denoted Δ G °' and refers to a process in which reactants and products have a concentration of 1.0 M in a solution with pH = 7.) At the same time, however, the reaction of water with ATP to yield ADP plus HOPO 3 2– is strongly favorable, with Δ G °' = –30.5 kJ/mol. When the two reactions are coupled, glucose reacts with ATP to yield glucose 6-phosphate plus ADP in a reaction that is favorable by about 16.7 kJ/mol (4.0 kcal/mol). That is, ATP drives the phosphorylation reaction of glucose. It’s this ability to drive otherwise unfavorable phosphorylation reactions that makes ATP so useful. The resultant phosphates are much more reactive as leaving groups in nucleophilic substitutions and eliminations than the alcohols they’re derived from and are therefore more chemically useful. Problems

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Catabolism of Triacylglycerols: The Fate of Glycerol The metabolic breakdown of triacylglycerols begins with their hydrolysis in the stomach and small intestine to yield glycerol plus fatty acids. The reaction is catalyzed by a lipase, whose mechanism is shown in . The active site of the enzyme contains a catalytic triad of aspartic acid, histidine, and serine residues, which act cooperatively to provide the necessary acid and base catalysis for the individual steps. Hydrolysis is accomplished by two sequential nucleophilic acyl substitution reactions, one that covalently binds an acyl group to the side chain –OH of a serine residue on the enzyme and a second that frees the fatty acid from the enzyme. MECHANISM Mechanism for the action of lipase. The active site of the enzyme contains a catalytic triad of aspartic acid, histidine, and serine, which react cooperatively to carry out two nucleophilic acyl substitution reactions. Individual steps are explained in the text. Steps 1 – 2 of : Acyl Enzyme Formation The first nucleophilic acyl substitution step—reaction of the triacylglycerol with the active-site serine to give an acyl enzyme—begins with deprotonation of the serine alcohol by histidine to form the more strongly nucleophilic alkoxide ion. This proton transfer is facilitated by a nearby side-chain carboxylate anion of aspartic acid, which makes the histidine more basic and stabilizes the resultant histidine cation by electrostatic interactions. The deprotonated serine adds to a carbonyl group of a triacylglycerol to give a tetrahedral intermediate, which expels a diacylglycerol as a leaving group and produces an acyl enzyme. This step is catalyzed by a proton transfer from histidine to make the leaving group a neutral alcohol. Steps 3 – 4 of : Hydrolysis The second nucleophilic acyl substitution step hydrolyzes the acyl enzyme and gives the free fatty acid by a mechanism analogous to that of the first two steps. Water is deprotonated by histidine to give hydroxide ion, which adds to the enzyme-bound acyl group. The tetrahedral intermediate then expels the neutral serine residue as the leaving group, freeing the fatty acid and returning the enzyme to its active form. The fatty acids released on triacylglycerol hydrolysis are transported to mitochondria and degraded to acetyl CoA, while the glycerol is carried to the liver for further metabolism. In the liver, glycerol is first phosphorylated by reaction with ATP and then oxidized by NAD + . The resulting dihydroxyacetone phosphate (DHAP) enters the carbohydrate glycolysis pathway, which we’ll discuss in . You might note that C2 of glycerol is a prochiral center ( ) with two identical “arms.” As is typical for enzyme-catalyzed reactions, the phosphorylation of glycerol is selective. Only the pro- R arm undergoes reaction, although this can’t be predicted in advance. Note also that the phosphorylation product is named sn -glycerol 3-phosphate, where the sn - prefix means “stereospecific numbering.” In this convention, the molecule is drawn as a Fischer projection with the − OH group at C2 pointing to the left and the glycerol carbon atoms numbered from the top.

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Catabolism of Triacylglycerols: β -Oxidation The fatty acids that result from triacylglycerol hydrolysis are converted into thioesters with coenzyme A and then catabolized by a repetitive four-step sequence of reactions called the β -oxidation pathway , shown in . Each passage along the pathway results in the cleavage of an acetyl group from the end of the fatty-acid chain, until the entire molecule is ultimately degraded. As each acetyl group is produced, it enters the citric acid cycle and is further degraded to CO 2 , as we’ll see in . MECHANISM The four steps of the β -oxidation pathway, resulting in the cleavage of an acetyl group from the end of the fatty-acid chain. The key chain-shortening step is a retro-Claisen reaction of a β -keto thioester. Individual steps are explained in the text. Step 1 of : Introduction of a Double Bond The β -oxidation pathway begins when two hydrogen atoms are removed from C2 and C3 of the fatty acyl CoA by one of a family of acyl-CoA dehydrogenases to yield an α , β -unsaturated acyl CoA. This kind of oxidation—the introduction of a conjugated double bond into a carbonyl compound—occurs frequently in biochemical pathways and usually involves the coenzyme flavin adenine dinucleotide (FAD). Reduced FADH 2 is the by-product. The mechanisms of FAD-catalyzed reactions are often difficult to establish because flavin coenzymes can follow both two-electron (polar) and one-electron (radical) pathways. As a result, extensive studies of the family of acyl-CoA dehydrogenases have not yet provided a clear picture of how these enzymes function. What is known is that: ( 1 ) The first step is abstraction of the pro- R hydrogen from the acidic α position of the acyl CoA to give a thioester enolate ion. Hydrogen-bonding between the acyl carbonyl group and the ribitol hydroxyls of FAD increases the acidity of the acyl group. ( 2 ) The pro- R hydrogen at the β position is transferred to FAD. ( 3 ) The α , β -unsaturated acyl CoA that results has a trans double bond. One suggested mechanism has the reaction taking place by a conjugate nucleophilic addition of hydride, analogous to what occurs during alcohol oxidations with NAD + . Electrons on the enolate ion might expel a β hydride ion, which could add to the doubly bonded N5 nitrogen on FAD. Protonation of the intermediate at N1 would give the product. Step 2 of : Conjugate Addition of Water The α , β -unsaturated acyl CoA produced in step 1 reacts with water by a conjugate addition pathway ( ) to yield a β -hydroxyacyl CoA in a process catalyzed by enoyl CoA hydratase. Water as nucleophile adds to the β carbon of the double bond, yielding an intermediate thioester enolate ion that is protonated on the α position. Step 3 of : Alcohol Oxidation The β -hydroxyacyl CoA from step 2 is oxidized to a β -ketoacyl CoA in a reaction catalyzed by one of a family of L-3-hydroxyacyl-CoA dehydrogenases, which differ in substrate specificity according to the chain length of the acyl group. As in the oxidation of sn -glycerol 3-phosphate to dihydroxyacetone phosphate mentioned at the end of , this alcohol oxidation requires NAD + as a coenzyme and yields reduced NADH/H + as by-product. Deprotonation of the hydroxyl group is carried out by a histidine residue at the active site. Step 4 of : Chain Cleavage Acetyl CoA is split off from the chain in the final step of β -oxidation, leaving an acyl CoA that is two carbon atoms shorter than the original. The reaction is catalyzed by β -ketoacyl-CoA thiolase and is mechanistically the reverse of a Claisen condensation reaction ( ). In the forward direction, a Claisen condensation joins two esters together to form a β -keto ester product. In the reverse direction, a retro-Claisen reaction splits apart a β -keto ester (or β -keto thioester in this case) to form two esters (or two thioesters). The retro-Claisen reaction occurs by nucleophilic addition of a cysteine −SH group on the enzyme to the keto group of the β -ketoacyl CoA to yield an alkoxide ion intermediate. Cleavage of the C2–C3 bond then follows, with the expulsion of an acetyl CoA enolate ion that is immediately protonated. The enzyme-bound acyl group then undergoes nucleophilic acyl substitution by reaction with a molecule of coenzyme A, and the chain-shortened acyl CoA that results re-enters the β -oxidation pathway for further degradation. Look at the catabolism of myristic acid shown in to see the overall results of the β -oxidation pathway. The first passage converts the 14-carbon myristoyl CoA into the 12-carbon lauroyl CoA plus acetyl CoA, the second passage converts lauroyl CoA into the 10-carbon caproyl CoA plus acetyl CoA, the third passage converts caproyl CoA into the 8-carbon capryloyl CoA, and so on. Note that the final passage produces two molecules of acetyl CoA because the precursor has four carbons. Catabolism of the 14-carbon myristic acid by the β -oxidation pathway yields seven molecules of acetyl CoA after six passages. Most fatty acids have an even number of carbon atoms, so none are left over after β -oxidation. Those fatty acids with an odd number of carbon atoms yield the three-carbon propionyl CoA in the final β -oxidation. Propionyl CoA is then converted to succinate by a multistep radical pathway, and succinate enters the citric acid cycle ( ). Note that the three-carbon propionyl group should technically be called propanoyl, but biochemists generally use the nonsystematic name. Problems

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Biosynthesis of Fatty Acids One of the most striking features of the common fatty acids is that they have an even number of carbon atoms ( ). This occurs because all fatty acids are derived biosynthetically from acetyl CoA by sequential addition of two-carbon units to a growing chain. The acetyl CoA, in turn, arises primarily from the metabolic breakdown of carbohydrates in the glycolysis pathway, which we’ll see in . Thus, dietary carbohydrates consumed in excess of immediate energy needs are turned into fats for storage. As a general rule in biological chemistry, the anabolic pathway by which a substance is made is not the reverse of the catabolic pathway by which the same substance is degraded. The two paths must differ in some respects for both to be energetically favorable. Thus, the β -oxidation pathway for converting fatty acids into acetyl CoA and the biosynthesis of fatty acids from acetyl CoA are related but are not exact opposites. Differences include the identity of the acyl-group carrier, the stereochemistry of the β -hydroxyacyl reaction intermediate, and the identity of the redox coenzyme. FAD is used to introduce a double bond in β -oxidation, while NADPH is used to reduce the double bond in fatty-acid biosynthesis. In bacteria, each step in fatty-acid synthesis is catalyzed by a separate enzyme. In vertebrates, however, fatty-acid synthesis is catalyzed by an immense, multienzyme complex called a synthase that contains two identical subunits of 2505 amino acids each and catalyzes all steps in the pathway. In fact, for an 18-carbon fatty acid, the synthase catalyzes 42 separate steps! An overview of fatty-acid biosynthesis is shown in . Steps 1 – 2 of : Acyl Transfers The starting material for fatty-acid biosynthesis is the thioester acetyl CoA, the final product of carbohydrate breakdown, as we’ll see in . The pathway begins with several priming reactions, which transport acetyl CoA and convert it into more reactive species. The first priming reaction is a nucleophilic acyl substitution reaction that converts acetyl CoA into acetyl ACP (acyl carrier protein). Notice that the mechanism of the nucleophilic acyl substitution in step 1 can be given in an abbreviated form that saves space by not explicitly showing the tetrahedral reaction intermediate. Instead, electron movement is shown as a heart-shaped path around the carbonyl oxygen to imply the two steps of the full mechanism. Biochemists commonly use this kind of abbreviated format, and we’ll also use it on occasion through the rest of this chapter. MECHANISM The pathway for fatty-acid biosynthesis from the two-carbon precursor, acetyl CoA. Individual steps are explained in the text. In bacteria, ACP is a small protein of 77 residues that transports an acyl group from one enzyme to another. In vertebrates, however, ACP appears to be a long arm on a multienzyme synthase complex, whose apparent function is to shepherd an acyl group from site to site within the complex. As in acetyl CoA, the acyl group in acetyl ACP is linked by a thioester bond to the sulfur atom of phosphopantetheine. The phosphopantetheine is in turn linked to ACP through the side-chain –OH group of a serine residue in the enzyme. Step 2 , another priming reaction, involves a further exchange of thioester linkages by another nucleophilic acyl substitution and results in covalent bonding of the acetyl group to a cysteine residue in the synthase complex that catalyzes the upcoming condensation step. Steps 3 – 4 of : Carboxylation and Acyl Transfer Step 3 is a loading reaction in which acetyl CoA is carboxylated by reaction with HCO 3 – and ATP to yield malonyl CoA plus ADP. This step requires the coenzyme biotin, which is bonded to the lysine residue of acetyl CoA carboxylase and acts as a carrier of CO 2 . Biotin first reacts with bicarbonate ion to give N -carboxybiotin, which then reacts with the enolate ion of acetyl CoA and transfers the CO 2 group. Thus, biotin acts as a carrier of CO 2 , binding it in one step and releasing it in another. The mechanism of the CO 2 transfer reaction with acetyl CoA to give malonyl CoA is thought to involve CO 2 as the reactive species. One proposal is that the loss of CO 2 is favored by hydrogen-bond formation between the N -carboxybiotin carbonyl group and a nearby acidic site in the enzyme. Simultaneous deprotonation of acetyl CoA by a basic site in the enzyme gives a thioester enolate ion that can react with CO 2 as it forms ( ). MECHANISM Mechanism of step 3 in , the biotin-dependent carboxylation of acetyl CoA to yield malonyl CoA. Following the formation of malonyl CoA, another nucleophilic acyl substitution reaction occurs in step 4 to form the more reactive malonyl ACP, thereby binding the malonyl group to an ACP arm of the multienzyme synthase. At this point, both acetyl and malonyl groups are bound to the enzyme, and the stage is set for their condensation. Step 5 of : Condensation The key carbon–carbon bond-forming reaction that builds the fatty-acid chain occurs in step 5 . This step is simply a Claisen condensation between acetyl synthase as the electrophilic acceptor and malonyl ACP as the nucleophilic donor. The mechanism of the condensation is thought to involve decarboxylation of malonyl ACP to give an enolate ion, followed by immediate nucleophilic addition of the enolate ion to the carbonyl group of acetyl synthase. Breakdown of the tetrahedral intermediate then gives the four-carbon condensation product acetoacetyl ACP and frees the synthase binding site for attachment of the chain-elongated acyl group at the end of the sequence. Steps 6 – 8 of : Reduction and Dehydration The ketone carbonyl group in acetoacetyl ACP is next reduced to the alcohol β -hydroxybutyryl ACP by β -keto thioester reductase and NADPH, a reducing coenzyme closely related to NADH. R stereochemistry results at the newly formed chirality center in the β -hydroxy thioester product. (Note that the systematic name of a butyryl group is butanoyl. ) Subsequent dehydration of β -hydroxybutyryl ACP by an E1cB reaction in step 7 yields trans -crotonyl ACP, and the carbon–carbon double bond of crotonyl ACP is reduced by NADPH in step 8 to yield butyryl ACP. The double-bond reduction occurs by conjugate nucleophilic addition of a hydride ion from NADPH to the β carbon of trans -crotonyl ACP. In vertebrates, this reduction occurs by an overall syn addition, but other organisms carry out similar chemistry with different stereochemistry. The net effect of the eight steps in the fatty-acid biosynthesis pathway is to take two 2-carbon acetyl groups and combine them into a 4-carbon butyryl group. Further condensation of the butyryl group with another malonyl ACP yields a 6-carbon unit, and still further repetitions add two carbon atoms at a time until the 16-carbon palmitoyl ACP is produced. Further chain elongation of palmitic acid occurs by reactions similar to those just described, but CoA rather than ACP acts as the carrier group, and separate enzymes are needed for each step rather than a multienzyme synthase complex. Problems

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Catabolism of Carbohydrates: Glycolysis Glucose is the body’s primary short-term energy source. Its catabolism begins with glycolysis , a series of ten enzyme-catalyzed reactions that break down glucose into 2 equivalents of pyruvate, CH 3 COCO 2 – . The steps of glycolysis, also called the Embden–Meyerhoff pathway after its discoverers, are summarized in . MECHANISM The ten-step glycolysis pathway for catabolizing glucose to two molecules of pyruvate. Individual steps are described in the text. MECHANISM (Continued) Steps 1 – 2 of : Phosphorylation and Isomerization Glucose, produced by the digestion of dietary carbohydrates, is phosphorylated at the C6 hydroxyl group by reaction with ATP in a process catalyzed by hexokinase. As noted in , the reaction requires Mg 2+ as a cofactor to complex with the negatively charged phosphate oxygens. The glucose 6-phosphate that results is then isomerized by glucose 6-phosphate isomerase to give fructose 6-phosphate. This isomerization takes place by initial opening of the glucose hemiacetal ring to its open-chain form, followed by keto–enol tautomerization to a cis enediol, HO ─ C ═ C ─ OH . But because glucose and fructose share a common enediol, further tautomerization to a different keto form produces open-chain fructose, and cyclization completes the process ( ). Mechanism of step 2 in glycolysis, the isomerization of glucose 6-phosphate to fructose 6-phosphate. Step 3 of : Phosphorylation Fructose 6-phosphate is converted in step 3 to fructose 1,6-bisphosphate (FBP) by a phosphofructokinase-catalyzed reaction with ATP (recall that the prefix bis - means two). The mechanism is similar to that in step 1, with Mg 2+ ion again required as cofactor. Interestingly, the product of step 2 is the α anomer of fructose 6-phosphate, but it is the β anomer that is phosphorylated in step 3, implying that the two anomers equilibrate rapidly through the open-chain form. The result is a molecule ready to be split into the two three-carbon intermediates that will ultimately become two molecules of pyruvate. Step 4 of : Cleavage Fructose 1,6-bisphosphate is cleaved in step 4 into two 3-carbon pieces, dihydroxyacetone phosphate (DHAP) and glyceraldehyde 3-phosphate (GAP). The bond between C3 and C4 of fructose 1,6-bisphosphate breaks, and a C ═ O group is formed at C4. Mechanistically, the cleavage is the reverse of an aldol reaction ( ) and is catalyzed by an aldolase. A forward aldol reaction joins two aldehydes or ketones to give a β -hydroxy carbonyl compound, while a retro-aldol reaction, as in this case, cleaves a β -hydroxy carbonyl compound into two aldehydes or ketones. Two classes of aldolases are used by organisms for catalysis of the retro-aldol reaction. In fungi, algae, and some bacteria, the retro-aldol reaction is catalyzed by class II aldolases, which function by coordination of the fructose carbonyl group with Zn 2+ as Lewis acid. In plants and animals, the reaction is catalyzed by class I aldolases and does not take place on the free ketone. Instead, fructose 1,6-bisphosphate undergoes reaction with the side-chain –NH 2 group of a lysine residue on the aldolase to yield a protonated enzyme-bound imine ( ), which is often called a Schiff base in biochemistry. Because of its positive charge, the iminium ion is a better electron acceptor than a ketone carbonyl group. Retro-aldol reaction ensues, giving glyceraldehyde 3-phosphate and an enamine, which is protonated to give another iminium ion that is hydrolyzed to yield dihydroxyacetone phosphate ( ). Mechanism of step 4 in , the cleavage of fructose 1,6-bisphosphate to yield glyceraldehyde 3-phosphate and dihydroxyacetone phosphate. The reaction occurs through an iminium ion formed by reaction with a lysine residue in the enzyme. Step 5 of : Isomerization Dihydroxyacetone phosphate is isomerized in step 5 by triose phosphate isomerase to form a second equivalent of glyceraldehyde 3-phosphate. As in the conversion of glucose 6-phosphate to fructose 6-phosphate in step 2 , the isomerization takes place by keto–enol tautomerization through a common enediol intermediate. A base deprotonates C1 and then reprotonates C2 using the same hydrogen. The net result of steps 4 and 5 together is the production of two glyceraldehyde 3-phosphate molecules, both of which pass down the rest of the pathway. Thus, each of the remaining five steps of glycolysis takes place twice for every glucose molecule entering at step 1. Steps 6 – 7 of : Oxidation, Phosphorylation, and Dephosphorylation Glyceraldehyde 3-phosphate is oxidized and phosphorylated in step 6 to give 1,3-bisphosphoglycerate ( ). The reaction is catalyzed by glyceraldehyde 3-phosphate dehydrogenase and begins by nucleophilic addition of the –SH group of a cysteine residue in the enzyme to the aldehyde carbonyl group to yield a hemithioacetal ( ), the sulfur analog of a hemiacetal. Oxidation of the hemithioacetal –OH group by NAD + then yields a thioester, which reacts with phosphate ion in a nucleophilic acyl substitution step to yield 1,3-bisphosphoglycerate, a mixed anhydride derived from a carboxylic acid and a phosphoric acid. Mechanism of step 6 in , the oxidation and phosphorylation of glyceraldehyde 3-phosphate to give 1,3-bisphosphoglycerate. The process occurs through initial formation of a hemiacetal that is oxidized to a thioester and converted into an acyl phosphate. Like all anhydrides ( ), the mixed carboxylic–phosphoric anhydride is a reactive substrate in nucleophilic acyl (or phosphoryl) substitution reactions. Reaction of 1,3-bisphosphoglycerate with ADP occurs in step 7 by substitution on phosphorus, resulting in transfer of a phosphate group to ADP and giving ATP plus 3-phosphoglycerate. The process is catalyzed by phosphoglycerate kinase and requires Mg 2+ as cofactor. Together, steps 6 and 7 accomplish the oxidation of an aldehyde to a carboxylic acid. Step 8 of : Isomerization 3-Phosphoglycerate isomerizes to 2-phosphoglycerate in a step catalyzed by phosphoglycerate mutase. In plants, 3-phosphoglycerate transfers its phosphoryl group from its C3 oxygen to a histidine residue on the enzyme in one step and then accepts the same phosphoryl group back onto the C2 oxygen in a second step. In animals and yeast, however, the enzyme contains a phosphorylated histidine, which transfers its phosphoryl group to the C2 oxygen of 3-phosphoglycerate and forms 2,3-bisphosphoglycerate as intermediate. The same histidine then accepts a phosphoryl group from the C3 oxygen to yield the isomerized product plus the regenerated enzyme. As explained in , we’ll occasionally use an abbreviated mechanism for nucleophilic acyl substitution reactions to save space. Steps 9 – 10 of : Dehydration and Dephosphorylation Like most β -hydroxy carbonyl compounds, 2-phosphoglycerate undergoes a ready dehydration in step 9 by an E1cB mechanism ( ). The process is catalyzed by enolase, and the product is phosphoenolpyruvate, abbreviated PEP. Two Mg 2+ ions are associated with the 2-phosphoglycerate to neutralize the negative charges. Transfer of the phosphoryl group to ADP in step 10 then generates ATP and gives enolpyruvate, which tautomerizes to pyruvate. The reaction is catalyzed by pyruvate kinase and requires that a molecule of fructose 1,6-bisphosphate also be present, as well as 2 equivalents of Mg 2+ . One Mg 2+ ion coordinates to ADP, and the other increases the acidity of a water molecule necessary for protonation of the enolate ion. The overall result of glycolysis can be summarized by the following equation: Problems

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Conversion of Pyruvate to Acetyl CoA Pyruvate, produced by catabolism of glucose (and by degradation of several amino acids), can undergo several further transformations depending on the conditions and on the organism. In the absence of oxygen, pyruvate can either be reduced by NADH to yield lactate [CH 3 CH(OH)CO 2 – ] or, in yeast, fermented to give ethanol. Under typical aerobic conditions in mammals, however, pyruvate is converted by a process called oxidative decarboxylation to give acetyl CoA plus CO 2 . ( Oxidative because the oxidation state of the carbonyl carbon rises from that of a ketone to that of a thioester.) The conversion occurs through a multistep sequence of reactions catalyzed by a complex of enzymes and cofactors called the pyruvate dehydrogenase complex. The process occurs in three stages, each catalyzed by one of the enzymes in the complex, as outlined in . Acetyl CoA, the ultimate product, then acts as fuel for the final stage of catabolism, the citric acid cycle. Step 1 of : Addition of Thiamin Diphosphate The conversion of pyruvate to acetyl CoA begins by reaction of pyruvate with thiamin diphosphate, a derivative of vitamin B 1 . Formerly called thiamin pyro phosphate, thiamin diphosphate is usually abbreviated as TPP. The spelling thiamine is also correct and frequently used. The key structural element in thiamin diphosphate is the thiazolium ring—a five-membered, unsaturated heterocycle containing a sulfur atom and a positively charged nitrogen atom. The thiazolium ring is weakly acidic, with a p K a of approximately 18 for the ring hydrogen between N and S. Bases can therefore deprotonate thiamin diphosphate, leading to formation of an ylide much like the phosphonium ylides used in Wittig reactions ( ). As in the Wittig reaction, the TPP ylide is a nucleophile and adds to the ketone carbonyl group of pyruvate to yield an alcohol addition product. MECHANISM Mechanism for the conversion of pyruvate to acetyl CoA through a multistep sequence of reactions that requires three different enzymes and four different coenzymes. The individual steps are explained in the text. Step 2 of : Decarboxylation The TPP addition product, which contains an iminium ion β to a carboxylate anion, undergoes decarboxylation in much the same way that a β -keto acid decarboxylates in the acetoacetic ester synthesis ( ). The C ═ N + bond of the pyruvate addition product acts like the C ═ O bond of a β -keto acid to accept electrons as CO 2 leaves, giving hydroxyethylthiamin diphosphate (HETPP). Step 3 of : Reaction with Lipoamide Hydroxyethylthiamin diphosphate is an enamine ( R 2 N ─ C ═ C ), which, like all enamines, is nucleophilic ( ). It therefore reacts with the enzyme-bound disulfide lipoamide by nucleophilic attack on a sulfur atom, displacing the second sulfur in an S N 2-like process. Step 4 of : Elimination of Thiamin Diphosphate The product of the HETPP reaction with lipoamide is a hemithioacetal, which eliminates thiamin diphosphate ylide. This elimination is the reverse of the ketone addition in step 1 and generates acetyl dihydrolipoamide. Step 5 of : Acyl Transfer Acetyl dihydrolipoamide, a thioester, undergoes a nucleophilic acyl substitution reaction with coenzyme A to yield acetyl CoA plus dihydrolipoamide. The dihydrolipoamide is then oxidized back to lipoamide by FAD ( ), and the FADH 2 that results is in turn oxidized back to FAD by NAD + , completing the catalytic cycle. Problems

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The Citric Acid Cycle The initial stages of catabolism result in the conversion of both fats and carbohydrates into acetyl groups that are bonded through a thioester link to coenzyme A. Acetyl CoA then enters the next stage of catabolism—the citric acid cycle , also called the tricarboxylic acid (TCA) cycle, or Krebs cycle, after Hans Krebs, who unraveled its complexities in 1937. The overall result of the cycle is the conversion of an acetyl group into two molecules of CO 2 plus reduced coenzymes by the eight-step reaction sequence shown in . As its name implies, the citric acid cycle is a closed loop of reactions in which the product of the final step (oxaloacetate) is a reactant in the first step. The intermediates are constantly regenerated, flowing continuously through the cycle, which operates as long as the oxidizing coenzymes NAD + and FAD are available. To meet this condition, the reduced coenzymes NADH and FADH 2 must be reoxidized via the electron-transport chain, which in turn relies on oxygen as the ultimate electron acceptor. Thus, the cycle is dependent on the availability of oxygen and on the operation of the electron-transport chain. MECHANISM The citric acid cycle is an eight-step series of reactions that results in the conversion of an acetyl group into two molecules of CO 2 plus reduced coenzymes. Individual steps are explained in the text. Step 1 of : Addition to Oxaloacetate Acetyl CoA enters the citric acid cycle in step 1 by nucleophilic addition to the oxaloacetate carbonyl group, to give ( S )-citryl CoA. This addition is an aldol reaction and is catalyzed by citrate synthase, as discussed in . ( S )-Citryl CoA is then hydrolyzed to citrate by a typical nucleophilic acyl substitution reaction with water, catalyzed by the same citrate synthase enzyme. Note that the hydroxyl-bearing carbon of citrate is a prochirality center and contains two identical arms. Because the initial aldol reaction of acetyl CoA to oxaloacetate occurs specifically from the Si face of the ketone carbonyl group, the pro- S arm of citrate is derived from acetyl CoA and the pro- R arm is derived from oxaloacetate. Step 2 of : Isomerization Citrate, a prochiral tertiary alcohol, is next converted into its isomer, (2 R ,3 S )-isocitrate, a chiral secondary alcohol. The isomerization occurs in two steps, both of which are catalyzed by the same aconitase enzyme. The initial step is an E1cB dehydration of a β -hydroxy acid to give cis -aconitate, the same sort of reaction that occurs in step 9 of glycolysis ( ). The second step is a conjugate nucleophilic addition of water to the C═C bond ( ). The dehydration of citrate takes place specifically on the pro- R arm—the one derived from oxaloacetate—rather than on the pro- S arm derived from acetyl CoA. Step 3 of : Oxidation and Decarboxylation (2 R ,3 S )-Isocitrate, a secondary alcohol, is oxidized by NAD + in step 3 to give the ketone oxalosuccinate, which loses CO 2 to give α -ketoglutarate. Catalyzed by isocitrate dehydrogenase, the decarboxylation is a typical reaction of a β -keto acid, just like that in the acetoacetic ester synthesis ( ). The enzyme requires a divalent cation as a cofactor to polarize the ketone carbonyl group and make it a better electron acceptor. Step 4 of : Oxidative Decarboxylation The transformation of α -ketoglutarate to succinyl CoA in step 4 is a multistep process just like the transformation of pyruvate to acetyl CoA that we saw in . In both cases, an α -keto acid loses CO 2 and is oxidized to a thioester in a series of steps catalyzed by a multienzyme dehydrogenase complex. As in the conversion of pyruvate to acetyl CoA, the reaction involves an initial nucleophilic addition reaction of thiamin diphosphate ylide to α -ketoglutarate, followed by decarboxylation. Reaction with lipoamide, elimination of TPP ylide, and finally a transesterification of the dihydrolipoamide thioester with coenzyme A yields succinyl CoA. Step 5 of : Acyl CoA Cleavage Succinyl CoA is converted to succinate in step 5 . This reaction is catalyzed by succinyl CoA synthetase and is coupled with phosphorylation of guanosine diphosphate (GDP) to give guanosine triphosphate (GTP). The overall transformation is similar to that of steps 6 through 8 in glycolysis ( ), in which a thioester is converted into an acyl phosphate and a phosphate group is then transferred to ADP. The overall result is a “hydrolysis” of the thioester group without involvement of water. Step 6 of : Dehydrogenation Succinate is dehydrogenated in step 6 by the FAD-dependent succinate dehydrogenase to give fumarate. This process is analogous to what occurs during the β -oxidation pathway of fatty-acid catabolism ( ). The reaction is stereospecific, removing the pro- S hydrogen from one carbon and the pro- R hydrogen from the other. Steps 7 – 8 of : Hydration and Oxidation The final two steps in the citric acid cycle are the conjugate nucleophilic addition of water to fumarate to yield ( S )-malate and the oxidation of ( S )-malate by NAD + to give oxaloacetate. The addition is catalyzed by fumarase and is mechanistically similar to the addition of water to cis -aconitate in step 2. This reaction occurs through an enolate-ion intermediate, which is protonated on the side opposite the OH, leading to a net anti addition. The final step is the oxidation of ( S )-malate by NAD + to give oxaloacetate, a reaction catalyzed by malate dehydrogenase. The citric acid cycle has now returned to its starting point, ready to revolve again. The overall result of the cycle is Problems

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Carbohydrate Biosynthesis: Gluconeogenesis Glucose is the body’s primary fuel when food is plentiful, but in times of fasting or prolonged exercise, glucose stores can become depleted. Most tissues then begin metabolizing fats as their source of acetyl CoA, but the brain is different. The brain relies almost entirely on glucose for fuel and is dependent on receiving a continuous supply in the blood. When the supply of glucose fails, even for a brief time, irreversible damage can occur. Thus, a pathway for synthesizing glucose from simple precursors is crucial. Higher organisms are not able to synthesize glucose from acetyl CoA but must instead use one of the three-carbon precursors lactate, glycerol, or alanine, all of which are readily converted into pyruvate. Pyruvate then becomes the starting point for gluconeogenesis , the 11-step biosynthetic pathway by which organisms make glucose ( ). The gluconeogenesis pathway is not the reverse of the glycolysis pathway by which glucose is degraded. As with the catabolic and anabolic pathways for fatty acids ( and ), the catabolic and anabolic pathways for carbohydrates differ in some details so that both are energetically favorable. MECHANISM The gluconeogenesis pathway for the biosynthesis of glucose from pyruvate. Individual steps are explained in the text. MECHANISM (Continued) Step 1 of : Carboxylation Gluconeogenesis begins with the carboxylation of pyruvate to yield oxaloacetate. The reaction is catalyzed by pyruvate carboxylase and requires ATP, bicarbonate ion, and the coenzyme biotin, which acts as a carrier to transport CO 2 to the enzyme active site. The mechanism is analogous to that of step 3 in fatty-acid biosynthesis ( ), in which acetyl CoA is carboxylated to yield malonyl CoA. Step 2 of : Decarboxylation and Phosphorylation Decarboxylation of oxaloacetate, a β -keto acid, occurs by the typical retro-aldol mechanism like that in step 3 in the citric acid cycle ( ), and phosphorylation of the resultant pyruvate enolate ion by GTP occurs concurrently to give phosphoenolpyruvate. This reaction is catalyzed by phosphoenolpyruvate carboxykinase. Steps 3 – 4 of : Hydration and Isomerization Conjugate nucleophilic addition of water to the double bond of phosphoenolpyruvate gives 2-phosphoglycerate by a process similar to that of step 7 in the citric acid cycle. Phosphorylation of C3 and dephosphorylation of C2 then yields 3-phosphoglycerate. Mechanistically, these steps are the reverse of steps 9 and 8 in glycolysis ( ), which have equilibrium constants near 1 so that substantial amounts of reactant and product are both present. Steps 5 – 7 of : Phosphorylation, Reduction, and Tautomerization Reaction of 3-phosphoglycerate with ATP generates the corresponding acyl phosphate, 1,3-bisphosphoglycerate, which binds to the glyceraldehyde 3-phosphate dehydrogenase by a thioester bond to a cysteine residue. Reduction of the thioester by NADH/H + yields the corresponding aldehyde, and keto–enol tautomerization of the aldehyde gives dihydroxyacetone phosphate. All three steps comprise a mechanistic reversal of the corresponding steps 7, 6, and 5 of glycolysis and have equilibrium constants near 1. Step 8 of : Aldol Reaction Dihydroxyacetone phosphate and glyceraldehyde 3-phosphate, the two 3-carbon units produced in step 7, join by an aldol reaction to give fructose 1,6-bisphosphate, the reverse of step 4 in glycolysis ( ). As in glycolysis, the reaction is catalyzed in plants and animals by a class I aldolase and takes place on an iminium ion formed by reaction of dihydroxyacetone phosphate with a side-chain lysine –NH 2 group on the enzyme. Loss of a proton from the neighboring carbon then generates an enamine, an aldol-like reaction ensues, and the product is hydrolyzed. Steps 9 – 10 of : Hydrolysis and Isomerization Hydrolysis of the phosphate group at C1 of fructose 1,6-bisphosphate gives fructose 6-phosphate. Although the result of the reaction is the exact opposite of step 3 in glycolysis, the mechanism is not. In glycolysis, phosphorylation is accomplished by reaction of fructose with ATP, with formation of ADP as by-product. The reverse of that process, however—the reaction of fructose 1,6-bisphosphate with ADP to give fructose 6-phosphate and ATP—is energetically unfavorable because ATP is too high in energy. Thus, an alternative pathway is used in which the C1 phosphate group is removed by a direct hydrolysis reaction, catalyzed by fructose 1,6-bisphosphatase. Following hydrolysis, keto–enol tautomerization of the carbonyl group from C2 to C1 gives glucose 6-phosphate. The isomerization is the reverse of step 2 in glycolysis. Step 11 of : Hydrolysis The final step in gluconeogenesis is the conversion of glucose 6-phosphate to glucose by a second phosphatase-catalyzed hydrolysis reaction. As just discussed for the hydrolysis of fructose 1,6-bisphosphate in step 9, and for the same energetic reasons, the mechanism of the glucose 6-phosphate hydrolysis is not the exact opposite of the corresponding step 1 in glycolysis. Interestingly, however, the mechanisms of the two phosphate hydrolysis reactions in steps 9 and 11 are not the same. In step 9, water is the nucleophile, but in the glucose 6-phosphate reaction of step 11, a histidine residue on the enzyme attacks phosphorus, giving a phosphoryl enzyme intermediate that subsequently reacts with water. The overall result of gluconeogenesis is summarized by the following equation: Problems

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Catabolism of Proteins: Deamination The catabolism of proteins is much more complex than that of fats and carbohydrates because each of the 20 α -amino acids is degraded through its own unique pathway. The general idea, however, is that ( 1 ) the α amino group is first removed as ammonia by a deamination process, ( 2 ) the ammonia is converted into urea, and ( 3 ) the remaining amino acid carbon skeleton (usually an α -keto acid) is converted into a compound that enters the citric acid cycle. Transamination Deamination is usually accomplished by a transamination reaction in which the –NH 2 group of the amino acid is exchanged with the keto group of α -ketoglutarate, forming a new α -keto acid plus glutamate. The overall process occurs in two parts, is catalyzed by aminotransferases, and involves participation of the coenzyme pyridoxal phosphate, abbreviated PLP, a derivative of pyridoxine (vitamin B 6 ). The aminotransferases differ in their specificity for amino acids, but the mechanism remains the same. The mechanism of the first part of transamination is shown in . The process begins with reaction between the α -amino acid and pyridoxal phosphate, which is covalently bonded to the aminotransferase by an imine linkage between the side-chain –NH 2 group of a lysine residue in the enzyme and the PLP aldehyde group. Deprotonation/reprotonation of the PLP–amino acid imine effects tautomerization of the imine C ═ N bond, and hydrolysis of the tautomerized imine gives an α -keto acid plus pyridoxamine phosphate (PMP). MECHANISM Mechanism for the enzyme-catalyzed, PLP-dependent transamination of an α -amino acid to give an α -keto acid. Individual steps are explained in the text. Step 1 of : Transimination The first step in trans amination is trans imination —the reaction of the PLP–enzyme imine with an α -amino acid to give a PLP–amino acid imine plus expelled enzyme as the leaving group. The reaction occurs by nucleophilic addition of the amino acid –NH 2 group to the C ═ N bond of the PLP imine, much as an amine adds to the C ═ O bond of a ketone or aldehyde in a nucleophilic addition reaction ( ). The protonated diamine intermediate undergoes a proton transfer and expels the lysine amino group in the enzyme to complete the step. Steps 2 – 4 of : Tautomerization and Hydrolysis Following formation of the PLP–amino acid imine in step 1 , a tautomerization of the C ═ N bond occurs in step 2 . The basic lysine residue in the enzyme that was expelled as a leaving group during transimination deprotonates the acidic α position of the amino acid, with the protonated pyridine ring of PLP acting as the electron acceptor. Reprotonation occurs on the carbon atom next to the ring, generating a tautomeric product that is the imine of an α -keto acid with pyridoxamine phosphate, abbreviated PMP. Hydrolysis of this PMP– α -keto acid imine then completes the first part of the transamination reaction. This is the mechanistic reverse of imine formation and occurs by nucleophilic addition of water to the imine, followed by proton transfer and expulsion of PMP as leaving group. Regeneration of PLP from PMP With PLP plus the α -amino acid now converted into PMP plus an α -keto acid, PMP must be transformed back into PLP to complete the catalytic cycle. The conversion occurs by another transamination reaction, this one between PMP and an α -keto acid, usually α -ketoglutarate. The products are PLP plus glutamate, and the mechanism is the exact reverse of that shown in . That is, PMP and α -ketoglutarate give an imine; the PMP– α -ketoglutarate imine undergoes tautomerization of the C ═ N bond to give a PLP–glutamate imine; and the PLP–glutamate imine reacts with a lysine residue on the enzyme in a transimination process to yield PLP–enzyme imine plus glutamate. Problems

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