Biochemistry, 4th Edition P81 potx

24.7 How Are Steroid Hormones Synthesized and Utilized? 763 Pregnenolone and Progesterone Are the Precursors of All Other Steroid Hormones Pregnenolone is transported from the mitochondria to the ER, where a hydroxyl oxidation and migration of the double bond yield progesterone. Pregnenolone syn- thesis in the adrenal cortex is activated by adrenocorticotropic hormone (ACTH), a peptide of 39 amino acid residues secreted by the anterior pituitary gland. Progesterone is secreted from the corpus luteum during the latter half of the menstrual cycle and prepares the lining of the uterus for attachment of a fertilized ovum. If an ovum attaches, progesterone secretion continues to ensure the suc- cessful maintenance of a pregnancy. Progesterone is also the precursor for synthe- sis of the other sex hormone steroids and the corticosteroids. Male sex hormone steroids are called androgens, and female hormones, estrogens. Testosterone is an androgen synthesized in males primarily in the testes (and in much smaller amounts in the adrenal cortex). Androgens are necessary for sperm maturation. Even nongonadal tissue (liver, brain, and skeletal muscle) is susceptible to the ef- fects of androgens. Testosterone is also produced primarily in the ovaries (and in much smaller amounts in the adrenal glands) of females as a precursor for the estrogens. ␤-Estradiol is the most prominent estrogen (Figure 24.44). O HO H 3 C H 3 C HO H 3 C H 3 C CH O H 3 C H 3 C O O H 3 C H 3 C O OH OH H 3 C HO H 3 C CH 2 OH O HO CH C O O CH 2 OH C O H 3 C H 3 C O OH HO Pregnenolone Cholesterol Desmolase (Mitochondria) Isocaproic aldehyde Progesterone (Endoplasmic reticulum) Testosterone ␤-Estradiol Aldosterone Cortisol FIGURE 24.44 The steroid hormones are synthesized from cholesterol, with intermediate formation of preg- nenolone and progesterone.Testosterone, the principal male sex hormone steroid, is a precursor to ␤-estradiol. Cortisol, a glucocorticoid, and aldosterone, a mineralo- corticoid, are also derived from progesterone. 764 Chapter 24 Lipid Biosynthesis Steroid Hormones Modulate Transcription in the Nucleus Steroid hormones act in a different manner from most hormones we have consid- ered. In many cases, they do not bind to plasma membrane receptors but rather pass easily across the plasma membrane. Steroids may bind directly to receptors in the nucleus or may bind to cytosolic steroid hormone receptors, which then enter the nucleus. In the nucleus, the hormone-receptor complex binds directly to spe- cific nucleotide sequences in DNA, increasing transcription of DNA to RNA (see Chapters 29 and 32). Cortisol and Other Corticosteroids Regulate a Variety of Body Processes Corticosteroids, including the glucocorticoids and mineralocorticoids, are made by the cortex of the adrenal glands on top of the kidneys. Cortisol (Figure 24.44) is rep- resentative of the glucocorticoids, a class of compounds that (1) stimulate gluco- neogenesis and glycogen synthesis in liver (by promoting the synthesis of PEP carboxykinase, fructose-1,6-bisphosphatase, glucose-6-phosphatase, and glycogen synthase); (2) inhibit protein synthesis and stimulate protein degradation in pe- ripheral tissues such as muscle; (3) inhibit allergic and inflammatory responses; (4) exert an immunosuppressive effect, inhibiting DNA replication and mitosis and repressing the formation of antibodies and lymphocytes; and (5) inhibit formation of fibroblasts involved in healing wounds and slow the healing of broken bones. Aldosterone, the most potent of the mineralocorticoids (Figure 24.44), is in- volved in the regulation of sodium and potassium balances in tissues. Aldosterone increases the kidney’s capacity to absorb Na ϩ , Cl Ϫ , and H 2 O from the glomerular fil- trate in the kidney tubules. Anabolic Steroids Have Been Used Illegally to Enhance Athletic Performance The dramatic effects of androgens on protein biosynthesis have led many athletes to the use of synthetic androgens, which go by the blanket term anabolic steroids. Despite numerous warnings from the medical community about side effects, which include kidney and liver disorders, sterility, and heart disease, abuse of such substances is epi- demic. Stanozolol (Figure 24.45) was one of the agents found in the blood and urine of Ben Johnson following his record-setting performance in the 100-meter dash in the 1988 Olympic Games. Because use of such substances is disallowed, Johnson lost his gold medal and Carl Lewis was declared the official winner. H 3 C H 3 C H OH CH 3 N HN Stanozolol FIGURE 24.45 The structure of stanozolol, an anabolic steroid. SUMMARY 24.1 How Are Fatty Acids Synthesized? The synthesis of fatty acids and other lipid components is different from their degradation. Fatty acid synthesis involves a set of reactions that follow a strategy different in several ways from the corresponding degradative process: 1. Intermediates in fatty acid synthesis are linked covalently to the sulf- hydryl groups of the acyl carrier proteins. In contrast, fatty acid break- down intermediates are bound to the OSH group of coenzyme A. 2. Fatty acid synthesis occurs in the cytosol, whereas fatty acid degrada- tion takes place in mitochondria. 3. In animals, the enzymes of fatty acid synthesis are components of one long polypeptide chain, the fatty acid synthase, whereas no sim- ilar association exists for the degradative enzymes. 4. The coenzyme for the oxidation–reduction reactions of fatty acid syn- thesis is NADP ϩ /NADPH, whereas degradation involves the NAD ϩ / NADH couple. 24.2 How Are Complex Lipids Synthesized? A common pathway oper- ates in nearly all organisms for the synthesis of phosphatidic acid, the pre- cursor to other glycerolipids. Glycerokinase catalyzes the phosphorylation of glycerol to form glycerol-3-phosphate, which is then acylated at both the 1- and 2-positions to yield phosphatidic acid. In eukaryotes, phosphatidic acid is converted directly either to diacylglycerol or to cytidine diphospho- diacylglycerol (or simply CDP-diacylglycerol). From these two precursors, all other glycerophospholipids in eukaryotes are derived. Phosphatidyl- ethanolamine synthesis begins with phosphorylation of ethanolamine to form phosphoethanolamine. The next reaction involves transfer of a cytidylyl group from CTP to form CDP-ethanolamine and pyrophosphate. Diacylglycerol then displaces CMP to form phosphatidylethanolamine. Eukaryotes also use CDP-diacylglycerol, derived from phosphatidic acid, as a precursor for several other important phospholipids, including phos- phatidylinositol (PI), phosphatidylglycerol (PG), and cardiolipin. 24.3 How Are Eicosanoids Synthesized, and What Are Their Functions? Eicosanoids are ubiquitous breakdown products of phospholipids. In re- sponse to appropriate stimuli, cells activate the breakdown of selected phospholipids. Phospholipase A 2 selectively cleaves fatty acids from the C-2 position of phospholipids. Often these are unsaturated fatty acids, Problems 765 among which is arachidonic acid. Arachidonic acid may also be released from phospholipids by the combined actions of phospholipase C (which yields diacylglycerols) and diacylglycerol lipase (which releases fatty acids). Animal cells can modify arachidonic acid and other poly- unsaturated fatty acids to produce so-called local hormones. These substances include the prostaglandins, as well as thromboxanes, leuko- trienes, and other hydroxyeicosanoic acids. 24.4 How Is Cholesterol Synthesized? The cholesterol biosynthetic pathway begins in the cytosol with the synthesis of mevalonate from acetyl-CoA. The first step is the condensation of two molecules of acetyl- CoA to form acetoacetyl-CoA. In the next reaction, acetyl-CoA and acetoacetyl-CoA join to form 3-hydroxy-3-methylglutaryl-CoA, which is abbreviated HMG-CoA, in a reaction catalyzed by HMG-CoA synthase. The third step in the pathway is the rate-limiting step in cholesterol biosynthesis; HMG-CoA undergoes two NADPH-dependent reductions to produce 3R-mevalonate. Biosynthesis of squalene involves conversion of mevalonate to isopentenyl pyrophosphate and dimethylallyl pyro- phosphate. Condensation of these two 5-carbon intermediates produces geranyl pyrophosphate; addition of another 5-carbon isopentenyl group gives farnesyl pyrophosphate. Both steps in the production of farnesyl pyrophosphate occur with release of pyrophosphate, hydrolysis of which drives these reactions forward. Two farnesyl pyrophosphates join to pro- duce squalene. Squalene monooxygenase converts squalene to squalene- 2,3-epoxide. A second ER membrane enzyme produces lanosterol, and another 20 steps are required to convert lanosterol to cholesterol. 24.5 How Are Lipids Transported Throughout the Body? Most lipids circulate in the body in the form of lipoprotein complexes. Simple, un- esterified fatty acids are merely bound to serum albumin and other pro- teins in blood plasma, but phospholipids, triacylglycerols, cholesterol, and cholesterol esters are all transported in the form of lipoproteins. At various sites in the body, lipoproteins interact with specific receptors and enzymes that transfer or modify their lipid cargoes. 24.6 How Are Bile Acids Biosynthesized? The formation of bile salts represents the major pathway for cholesterol degradation. The first step involves hydroxylation at C-7. 7␣-Hydroxylase is a mixed-function oxi- dase involving cytochrome P-450. Mixed-function oxidases use O 2 as substrate. One oxygen atom goes to hydroxylate the substrate while the other is reduced to water. The function of cytochrome P-450 is to acti- vate O 2 for the hydroxylation reaction. Such hydroxylations are quite common in the synthetic routes for cholesterol, bile acids, and steroid hormones and also in detoxification pathways for aromatic compounds. 24.7 How Are Steroid Hormones Synthesized and Utilized? Biosyn- thesis of steroid hormones begins with the desmolase reaction, which converts cholesterol to pregnenolone. Desmolase activity includes two hydroxylases and utilizes cytochrome P-450. Pregnenolone is trans- ported from the mitochondria to the ER, where a hydroxyl oxidation and migration of the double bond yield progesterone. Progesterone is also the precursor for synthesis of the sex hormone steroids and the cor- ticosteroids. Testosterone is an androgen synthesized in males primarily in the testes. ␤-Estradiol is the most prominent estrogen. Aldosterone, the most potent of the mineralocorticoids, is involved in the regulation of sodium and potassium balances in tissues. PROBLEMS Preparing for an exam? Create your own study path for this chapter at www.cengage.com/login. 1. Carefully count and account for each of the atoms and charges in the equations for the synthesis of palmitoyl-CoA, the synthesis of malonyl-CoA, and the overall reaction for the synthesis of palmitoyl- CoA from acetyl-CoA. 2. (Integrates with Chapters 18 and 19.) Use the relationships shown in Figure 24.1 to determine which carbons of glucose will be in- corporated into palmitic acid. Consider the cases of both citrate that is immediately exported to the cytosol following its synthesis and citrate that enters the TCA cycle. 3. Based on the information presented in the text and in Figures 24.4 and 24.5, suggest a model for the regulation of acetyl-CoA carboxy- lase. Consider the possible roles of subunit interactions, phospho- rylation, and conformation changes in your model. 4. Consider the role of the pantothenic acid groups in animal FAS and the size of the pantothenic acid group itself, and estimate a maximal separation between the malonyl transferase and the ␤-ketoacyl-ACP synthase active sites. 5. Carefully study the reaction mechanism for the stearoyl-CoA desat- urase in Figure 24.14, and account for all of the electrons flowing through the reactions shown. Also account for all of the hydrogen and oxygen atoms involved in this reaction, and convince yourself that the stoichiometry is correct as shown. 6. Write a balanced, stoichiometric reaction for the synthesis of phos- phatidylethanolamine from glycerol, fatty acyl-CoA, and ethanol- amine. Make an estimate of the ⌬G°Ј for the overall process. 7. Write a balanced, stoichiometric reaction for the synthesis of cho- lesterol from acetyl-CoA. 8. Trace each of the carbon atoms of mevalonate through the synthe- sis of cholesterol, and determine the source (that is, the position in the mevalonate structure) of each carbon in the final structure. 9. Suggest a structural or functional role for the O -linked saccharide domain in the LDL receptor (Figure 24.40). 10. Identify the lipid synthetic pathways that would be affected by ab- normally low levels of CTP. 11. Determine the number of ATP equivalents needed to form palmitic acid from acetyl-CoA. (Assume for this calculation that each NADPH is worth 3.5 ATPs.) 12. Write a reasonable mechanism for the 3-ketosphinganine synthase reaction, remembering that it is a pyridoxal phosphate–dependent reaction. 13. Why is the involvement of FAD important in the conversion of stearic acid to oleic acid? 14. Write a suitable mechanism for the HMG-CoA synthase reaction. What is the chemistry that drives this condensation reaction? 15. Write a suitable reaction mechanism for the ␤-ketoacyl-ACP syn- thase, showing how the involvement of malonyl-CoA drives this reaction forward. 16. In the FAS megasynthase structures, the multiple functional sites must lie within reach of the ACP and its bound acyl group sub- strates. Examine the mammalian FAS structure (see Figure 24.11) and determine the distances between the various functional sites. You could approach this problem either by using a molecular mod- eling program (such as PyMol at www.pymol.org) or by consulting appropriate references (the following end-of-chapter reference is a good place to start: Maier, T., Leibundgut, M., and Ban, N., 2008. Science 321:1315–1322). You should convince yourself, with quanti- tative arguments, that the intersite distances can be traversed ap- propriately by the ACP group. 17. In the LDL receptor structure shown in Figure 24.41c, the ␤-propellor interaction with domains R4 and R5 is partly stabilized by salt bridges between acidic residues on R4 and R5 that also coordinate Ca 2ϩ ions. Use a molecular modeling program or con- sult the literature to identify at least two such interactions. Two suit- able references are Beglova, N., and Blacklow, S. C., 2005. Trends in Biochemical Sciences 30:309–316; and Rudenko, G., Henry, L., et al., 2002. Science 298:2353–2358. 766 Chapter 24 Lipid Biosynthesis 18. Insights into the function of LDL receptors in displacing LDL par- ticles in endosomes have come from an unlikely source: a study of LDL receptor binding by a human rhinovirus HRV2 (a common cold virus). Consult suitable references to learn how this study pro- vided support for the model of LDL particle displacement pre- sented in this chapter. Good references are Blacklow, S. C., 2004. Nature Structural and Molecular Biology 11:388–390; Verdaguer, N., Fita, I., et al., 2004. Nature Structural and Molecular Biology 11: 429–434; and Beglova, N., and Blacklow, S. C., 2005. Trends in Bio- chemical Sciences 30:309–316. Preparing for the MCAT Exam 19. Consider the synthesis of linoleic acid from palmitic acid and iden- tify a series of three consecutive reactions that embody chemistry similar to three reactions in the tricarboxylic acid cycle. 20. Rewrite the equation in Section 24.1 to describe the synthesis of be- henic acid (see Table 8.1). FURTHER READING General Chun, J., 2007. The sources of a lipid conundrum. Science 316:208–210. Lusis, A., and Pajukanta, P., 2008. A treasure trove for lipoprotein biol- ogy. Nature Genetics 40:129–130. Ohlrogge, J., and Browse, J., 1995. Lipid biosynthesis. Plant Cell 7:957–970. Smith, W. L., 2007. Nutritionally essential fatty acids and biologically in- dispensable cyclooxygenases. Trends in Biochemical Sciences 33:27–37. Vance, D. E., and Vance, J. E., 2008. Biochemistry of Lipids, Lipoproteins and Membranes, 5th ed., Amsterdam: Elsevier. Wolfgang, M. J., and Lane, M. D., 2006. The role of hypothalamic malonyl-CoA in energy homeostasis. Journal of Biological Chemistry 281:37265–37269. Acetyl-CoA Carboxylase Bilder, P., Lightle, S., et al., 2006. The structure of the carboxyltrans- ferase component of acetyl-CoA carboxylase reveals a zinc-binding motif unique to the bacterial enzyme. Biochemistry 45:1712–1722. Cho, Y. S., Lee, J. I., et al., 2007. Crystal structure of the biotin carboxy- lase domain of human acetyl-CoA carboxylase 2. Proteins 70: 268–272. Munday, M. R., 2002. Regulation of mammalian acetyl-CoA carboxylase. Biochemical Society Transactions 30:1059–1064. Tong, L., 2005. Acetyl-coenzyme A carboxylase: Crucial metabolic en- zyme and attractive target for drug discovery. Cellular and Molecular Life Sciences 62:1784–1803. Tong, L., and Harwood, H. J., Jr., 2006. Acetyl-coenzyme A carboxylases: Versatile targets for drug discovery. Journal of Cellular Biochemistry 99:1476–1488. Zhang, H., Tweel, B., et al., 2004a. Crystal structure of the carboxyl- transferase domain of acetyl-coenzyme A carboxylase in complex with CP-640186. Structure 12:1683–1691. Zhang, H., Tweel, B., et al., 2004b. Molecular basis for the inhibition of the carboxyltransferase domain of acetyl-coenzyme A carboxylase by haloxyfop and diclofop. Proceedings of the National Academy of Sciences U.S.A. 101:5910–5915. Zhang, H., Yang, Z., et al., 2003. Crystal structure of the carboxyl- transferase domain of acetyl-coenzyme A carboxylase. Science 299: 2064–2067. Fatty Acid Metabolism Asturias, F. J., Chadick, J. Z., et al., 2005. Structure and molecular orga- nization of mammalian fatty acid synthase. Nature Structural and Molecular Biology 12:225–232. Jakobsson, A., Westerberg, R., et al., 2006. Fatty acid elongases in mam- mals: Their regulation and roles in metabolism. Progress in Lipid Re- search 45:237–249. Jenni, S., Leibundgut, M., et al., 2007. Structure of fungal fatty acid syn- thase and implications for iterative substrate shuttling. Science 316: 254–261. Jump, D. B., 2002. The biochemistry of n-3 polyunsaturated fatty acids. Journal of Biological Chemistry 277:8755–8758. Kim, H Y., 2007. Novel metabolism of docosahexaenoic acid in neural cells. Journal of Biological Chemistry 282:18661–18665. Kresge, N., Simoni, R. D., et al., 2006. Salih Wakil’s elucidation of the animal fatty acid synthetase complex architecture. Jour nal of Biologi- cal Chemistry 281:e5–e7. Leibundgut, M., Jenni, S., et al., 2007. Structural basis for substrate de- livery by acyl carrier protein in the yeast fatty acid synthase. Science 316:288–290. Maier, T., Jenni, S., and Ban, N., 2006. Architecture of mammalian fatty acid synthase at 4.5Å resolution. Science 311:1258–1262. Maier, T., Leibundgut, M., and Ban, N., 2008. The crystal structure of a mammalian fatty acid synthase. Science 321:1315–1322. Reshef, L., Olswang, Y., et al., 2003. Glyceroneogenesis and the trigly- ceride/fatty acid cycle. Journal of Biological Chemistry 278:30413–30416. Riezman, H., 2007. The long and short of fatty acid synthesis. Cell 130: 587–588. Simard, J. R., Zunszain, P. A., et al., 2005. Locating high-affinity fatty acid–binding sites on albumin by X-ray crystallography and NMR spectroscopy. Proceedings of the National Academy of Sciences U.S.A. 102: 17958–17963. Smith, S., and Tsai, S-C., 2007. The type I fatty acid and polyketide syn- thases: A tale of two megasynthases. Natural Product Reports 24: 1041–1072. White, S. W., Zheng, J., et al., 2005. The structural biology of type II fatty acid biosynthesis. Annual Review of Biochemistry 74:791–831. Zhang, Y M., White, S. W., et al., 2006. Inhibiting bacterial fatty acid syn- thesis. Journal of Biological Chemistry 281:17541–17544. Function and Synthesis of Eicosanoids and Essential Fatty Acids Grosser, T., Fries, S., et al., 2006. Biological basis for the cardiovascular consequences of COX-2 inhibition: Therapeutic challenges and op- portunities. Journal of Clinical Nutrition 116:4–15. Hunter, W. N., 2007. The non-mevalonate pathway of isoprenoid pre- cursor biosynthesis. Journal of Biological Chemistry 282:21573–21577. Kresge, N., Simoni, R. D., et al., 2006. The prostaglandins, Sune Bergström and Bengt Samuelsson. Journal of Biological Chemistry 281:e9–e11. Kurumbail, R. G., Stevens, A. M., et al., 1996. Structural basis for selec- tive inhibition of cyclooxygenase-2 by anti-inflammatory agents. Na- ture 384:644–648. Lands, W. E., 1991. Biosynthesis of prostaglandins. Annual Review of Nu- trition 11:41–60. Malkowski, M. G., Thuresson, E. D., et al., 2001. Structure of eicosapen- taenoic and linoleic acids in the cyclooxygenase site of prosta- glandin endoperoxide H synthase-1. Journal of Biological Chemistry 276:37547–37555. Marszalek, J. R., and Lodish, H. F., 2005. Docosahexaenoic acid, fatty acid–interacting proteins, and neuronal function: Breastmilk and fish are g ood for you. Annual Review of Cell and Developmental Biology 21:633–657. Smith, W. L., 2007. Nutritionally essential fatty acids and biologically in- dispensable cyclooxygenases. Trends in Biochemical Sciences 33:27–37. Sugimoto, Y., and Narumiya, S., 2007. Prostaglandin E receptors. Jour- nal of Biological Chemistry 282:11613–11617. Further Reading 767 Phospholipid and Triacylglycerol Synthesis Carman, G. M., and Henry, S. A., 1989. Phospholipid biosynthesis in yeast. Annual Review of Biochemistry 58:635–669. Carman, G. M., and Henry, S A., 2007. Phosphatidic acid plays a central role in the transcriptional regulation of glycerophospholipid syn- thesis in Saccharomyces cerevisiae. Journal of Biological Chemistry 282: 37293–37297. Dunne, S. J., Cornell, R. B., et al., 1996. Structure of the membrane- binding domain of CTP phosphocholine cytidylyltransferase. Bio- chemistry 35:11975–11984. Han, G S., Wu, W I., et al., 2006. The Saccharomyces cerevisiae Lipin ho- molog is a Mg 2ϩ -dependent phosphatidate enzyme. Journal of Bio- logical Chemistry 281:9210–9218. Jackowski, S., 1996. Cell cycle regulation of membrane phospholipid metabolism. Journal of Biological Chemistry 271:20219–20222. Sohlencamp, C., Lopez-Lara, I. M., et al., 2003. Biosynthesis of phos- phatidylcholine in bacteria. Progress in Lipid Research 42:115–162. Sorger, D., and Daum, G., 2003. Triacylglycerol biosynthesis in yeast. Ap- plied Microbiology and Biotechnology 61:289–299. Tafesse, F. G., Ternes, P., et al., 2006. The multigenic sphingomyelin syn- thase family. Journal of Biological Chemistry 281:29421–29425. Vance, D. E., Li, Z., et al., 2007. Hepatic phosphatidylethanolamine N-methyltransferase, unexpected roles in animal biochemistry and physiology. Journal of Biological Chemistry 282:33237–33241. Watkins, P. A., 2008. Very long-chain acyl-CoA synthetases. Journal of Biological Chemistry 283:1773–1777. Structure and Function of Lipoproteins and Their Receptors Beglova, N., and Blacklow, S. C., 2005. The LDL receptor: How acid pulls the trigger. Trends in Biochemical Sciences 30:309–316. Blacklow, S. C., 2004. Catching the common cold. Nature Structural and Molecular Biology 11:388–390. Davidson, W. S., and Thompson, T. B., 2007. The structure of apolipo- protein A-1 in high density lipoproteins. Journal of Biological Chem- istry 282:22249–22253. Innerarity, T. L., 2002. LDL receptor’s ␤-propeller displaces LDL. Science 298:2337–2338. Johs, A., Hammel, M., et al., 2006. Modular structure of solubilized human apolipoprotein B-100. Journal of Biological Chemistry 281: 19732–19739. Rudenko, G., Henry, L., et al., 2002. Structure of the LDL receptor ex- tracellular domain at endosomal pH. Science 298:2353–2358. Verdaguer, N., Fita, I., Reithmayer, M., Moser, R., and Blaas, D., 2004. X-ray structure of a minor group human rhinovirus bound to a frag- ment of its cellular receptor protein. Nature Structural and Molecular Biology 11:429–434. Cholesterol Metabolism Bloch, K., 1965. The biological synthesis of cholesterol. Science 150: 19–28. Bloch, K., 1987. Summing up. Annual Review of Biochemistry 56:1–19. Bouvier, F., Rahier, A., et al., 2005. Biogenesis, molecular regulation and function of plant isoprenoids. Progress in Lipid Research 44:357–429. Brown, M. S., and Goldstein, J. L., 2006. Lowering LDL: Not only how low, but how long? Science 311:1721–1723. Dietschy, J. M., and Turley, S. D., 2002. Control of cholesterol turnover in the mouse. Journal of Biological Chemistry 277:3801–3804. Edwards, P. A., and Ericsson, J., 1999. Sterols and isoprenoids: Signaling molecules derived from the cholesterol biosynthetic pathway. An- nual Review of Biochemistry 68:157–185. Gimpl, G., Burger , K., et al., 2002. A closer look at the cholesterol sen- sor. Trends in Biochemical Sciences 27:596–599. Goldstein, J. L., and Brown, M. S., 2001. The cholesterol quartet. Science 292:1310–1312. Istvan, E. S., and Deisenhofer, J., 2001. Structural mechanism for statin inhibition of HMG-CoA reductase. Science 292:1160–1164. Kresge, N., Simoni, R. D., et al., 2006. 30 years of cholesterol metabo- lism: The work of Michael Brown and Joseph Goldstein. Journal of Biological Chemistry 281:e25–e28. © Royalty-Free/CORBIS 25 Nitrogen Acquisition and Amino Acid Metabolism Amino acids and nucleotides, as well as their polymeric forms (proteins and nucleic acids), are nitrogen-containing molecules upon which cell structure and function rely. How do these various organic forms of nitrogen arise? As we look at these com- pounds, an obvious feature is that nitrogen atoms are typically bound to carbon and/or hydrogen atoms. That is, the nitrogen atom is in a reduced state. On the other hand, the prevalent forms of nitrogen in the environment are inorganic and oxidized; N 2 (dinitrogen gas) and NO 3 Ϫ (nitrate ions) being the principal species. The two principal routes for nitrogen acquisition from the inanimate environment, nitrate assimilation and nitrogen fixation, lead to formation of ammonium ions (NH 4 ϩ ). Reactions that incorporate NH 4 + into organic linkage (the reactions of am- monium assimilation) follow. Among these, glutamine synthetase merits particular attention because it conveys several important lessons in metabolic regulation. This chapter presents the pathways of amino acid biosynthesis and degradation; those in- volving the sulfur-containing amino acids provide an opportunity to introduce as- pects of sulfur metabolism. 25.1 Which Metabolic Pathways Allow Organisms to Live on Inorganic Forms of Nitrogen? Nitrogen Is Cycled Between Organisms and the Inanimate Environment Nitrogen acquisition by biological systems is accompanied by its reduction to am- monium ion (NH 4 ϩ ) and the incorporation of NH 4 ϩ into organic linkage as amino or amido groups (Figure 25.1). The reduction of NO 3 Ϫ to NH 4 ϩ occurs in green plants, various fungi, and certain bacteria in a two-step metabolic pathway known as nitrate assimilation. The formation of NH 4 ϩ from N 2 gas is termed nitrogen fix- ation. N 2 fixation is an exclusively prokaryotic process, although bacteria in symbi- otic association with certain green plants also carry out nitrogen fixation. No ani- mals are capable of either nitrogen fixation or nitrate assimilation, so they are totally dependent on plants and microorganisms for the synthesis of organic ni- trogenous compounds, such as amino acids and proteins, to satisfy their require- ments for this essential element. Animals release excess nitrogen in a reduced form, either as NH 4 ϩ or as organic nitrogenous compounds such as urea. The release of N occurs both during life and as a consequence of microbial decomposition following death. Various bacteria re- turn the reduced forms of nitrogen back to the environment by oxidizing them. The oxidation of NH 4 ϩ to NO 3 Ϫ by nitrifying bacteria, a group of chemoau- totrophs, provides the sole source of chemical energy for the life of these microbes. Nitrate nitrogen also returns to the atmosphere as N 2 as a result of the metabolic Soybeans. Only plants and certain microorganisms are able to transform the oxidized, inorganic forms of nitrogen available in the inanimate environment into reduced, biologically useful forms. Soybean plants can meet their nitrogen requirements both by assimilat- ing nitrate and, in symbiosis with bacteria, fixing N 2 . I was determined to know beans. Henry David Thoreau (1817–1862) The Writings of Henry David Thoreau, vol. 2, p. 178, Houghton Mifflin (1906) KEY QUESTIONS 25.1 Which Metabolic Pathways Allow Organisms to Live on Inorganic Forms of Nitrogen? 25.2 What Is the Metabolic Fate of Ammonium? 25.3 What Regulatory Mechanisms Act on Escherichia coli Glutamine Synthetase? 25.4 How Do Organisms Synthesize Amino Acids? 25.5 How Does Amino Acid Catabolism Lead into Pathways of Energy Production? ESSENTIAL QUESTIONS Nitrogen is an essential nutrient for all cells.Amino acids provide nitrogen for the synthesis of other nitrogen-containing biomolecules. Excess amino acids in the diet can be converted into ␣-keto acids and used for energy production. What are the biochemical pathways that form ammonium from inorganic nitro- gen compounds prevalent in the inanimate environment? How is ammonium incorporated into organic compounds? How are amino acids synthesized and degraded? Create your own study path for this chapter with tutorials, simulations, animations, and Active Figures at www.cengage.com/login. 25.1 Which Metabolic Pathways Allow Organisms to Live on Inorganic Forms of Nitrogen? 769 activity of denitrifying bacteria. These bacteria are capable of using NO 3 Ϫ and sim- ilar oxidized inorganic forms of nitrogen as electron acceptors in place of O 2 in energy-producing pathways. The NO 3 Ϫ is reduced ultimately to dinitrogen (N 2 ). These bacteria thus deplete the levels of combined nitrogen, that is, N joined with other elements in chemical compounds. Combined nitrogen is important as nat- ural fertilizer. However, the denitrifying activity of bacteria is exploited in water treatment plants to reduce the load of combined nitrogen that might otherwise enter lakes, streams, and bays. Nitrate Assimilation Is the Principal Pathway for Ammonium Biosynthesis Nitrate assimilation occurs in two steps: the two-electron reduction of nitrate to ni- trite, catalyzed by nitrate reductase (Equation 25.1), followed by the six-electron reduction of nitrite to ammonium, catalyzed by nitrite reductase (Equation 25.2). (1) NO 3 Ϫ ϩ 2 H ϩ ϩ 2 e Ϫ ⎯⎯→NO 2 Ϫ ϩ H 2 O (25.1) (2) NO 2 Ϫ ϩ 8 H ϩ ϩ 6 e Ϫ ⎯⎯→NH 4 ϩ ϩ 2 H 2 O (25.2) Nitrate assimilation is the predominant means by which green plants, algae, and many microorganisms acquire nitrogen. The pathway of nitrate assimilation ac- counts for more than 99% of the inorganic nitrogen (nitrate or N 2 ) assimilated into organisms. Nitrate Reductase Contains Cytochrome b 557 and Molybdenum Cofactor A pair of electrons is transferred from NADH via enzyme-associated sulfhydryl groups, FAD, cytochrome b 557 , and MoCo (an essential molybdenum-containing cofactor) to nitrate, reducing it to nitrite. The brackets [ ] denote the protein- bound prosthetic groups that constitute an e Ϫ transport chain between NADH and nitrate. Nitrate reductases typically are cytosolic 220-kD dimeric proteins. The structure of the molybdenum cofactor (MoCo) is shown in Figure 25.2a. Molybdenum cofactor is necessary for both nitrate reductase activity and the as- sembly of nitrate reductase subunits into the active dimeric holoenzyme form. Molybdenum cofactor is also an essential cofactor for a variety of enzymes that catalyze hydroxylase-type reactions, including xanthine dehydrogenase, aldehyde oxidase, and sulfite oxidase. (25.3) NADH NADH ϩ NO 3 Ϫ NO 2 Ϫ [ SH FAD cytochrome b 557 MoCo] N 2 Organic N NH + 4 NO – 2 NO – 3 NO – 2 NO N 2 O Nitrate respiration (dissimilation) N i t r o g e n f i x a t i o n a s s i m i l a t i o n N i t r a t e N i t r i f i c a t i o n D e n i t r i f i c a t i o n Aerobic Anaerobic FIGURE 25.1 The nitrogen cycle. Organic nitrogenous compounds are formed by the incorporation of NH 4 ϩ into carbon skeletons. Note that denitrification and nitrogen fixation are anaerobic processes. Corn nitrate reductase cytochrome domain (FAD shown in yellow) (pdb id = 1CNE) Fungal nitrate reductase molybdenum cofactor domain (Mo cofactor in gold) ( p db id = 2BIH) Nitrate reductase has two structural domains, the cyto- chrome b domain (top) and the molybdenum cofactor domain (bottom). 770 Chapter 25 Nitrogen Acquisition and Amino Acid Metabolism Nitrite Reductase Contains Siroheme Six electrons are required to reduce NO 2 Ϫ to NH 4 ϩ . Nitrite reductases in photosynthetic organisms obtain these electrons from six molecules of photosynthetically reduced ferredoxin (Fd red ). Photosynthetic nitrite reductases are 63-kD monomeric proteins having a tetranuclear iron–sulfur cluster and a novel heme, termed siroheme, as pros- thetic groups. The [4Fe-4S] cluster and the siroheme act as a coupled e Ϫ transfer center. Nitrite binds directly to siroheme, providing the sixth ligand, much as O 2 binds to the heme of hemoglobin. Nitrite is reduced to ammonium while lig- anded to siroheme. The structure of siroheme is shown in Figure 25.2b. In higher plants, nitrite reductase is found in chloroplasts, where it has ready ac- cess to its primary reductant, photosynthetically reduced ferredoxin. Microbial ni- trite reductases closely resemble nitrate reductases in having essential OSH groups and FAD prosthetic groups to couple enzyme-mediated NADPH oxidation to nitrite reduction (Figure 25.3). (25.4) Light 6 Fd red 6 Fd ox NO 2 Ϫ NH 4 ϩ [(4Fe-4S) siroheme] COOH NN NN Fe COOH COOH COOH COOH H 3 C COOH HOOC H 3 C HOOC O H 2 N HN NN H H N C C SS Mo OO CHOH CH 2 OPO 3 2 – (a) (b) FIGURE 25.2 The novel prosthetic groups of nitrate reductase and nitrite reductase. (a) The molybdenum cofactor of nitrate reductase. (b) Siroheme, an essential prosthetic group of nitrite reductase. Siroheme is novel among hemes in having eight carboxylate-containing side chains. These carboxylate groups may act as H ϩ donors during the reduction of NO 2 Ϫ to NH 4 ϩ . Sequence Organization of the Nitrate Assimilation Enzymes Plant and Fungal Nitrate Reductases (~200-kD homodimers) N-term MoCo/NO 3 – hinge cytochrome b hinge FAD NAD(P)H 1 112 482 542 620 656 787 917 Plant Nitrite Reductases (63-kD monomers) e – donor FeS-siroheme/NO 2 – 473 518 566 Fungal Nitrite Reductases (~250-kD homodimers) FAD NAD(P)H Cys-rich FeS-siroheme/NO 2 – 26 60 183 215 496 600 715 763 1176 FIGURE 25.3 Domain organization within the enzymes of nitrate assimilation.The numbers denote residue number along the amino acid sequence of the proteins. Spinach nitrite reductase (iron-sulfur cluster in gold, siroheme in red) ( p db id = 2AKJ) 25.1 Which Metabolic Pathways Allow Organisms to Live on Inorganic Forms of Nitrogen? 771 Organisms Gain Access to Atmospheric N 2 Via the Pathway of Nitrogen Fixation Nitrogen fixation involves the reduction of nitrogen gas (N 2 ) via an enzyme system found only in prokaryotic cells. The heart of the nitrogen fixation process is the en- zyme known as nitrogenase, which catalyzes the reaction N 2 ϩ 10 H ϩ ϩ 8 e Ϫ ⎯⎯→2 NH 4 ϩ ϩ H 2 (25.5) Note that an obligatory reduction of two protons to hydrogen gas accompanies the biological reduction of N 2 to ammonia. Less than 1% of the inorganic N incorpo- rated into organic compounds by organisms can be attributed to nitrogen fixation; however, this process is the only way that organisms can tap into the enormous reservoir of N 2 in the atmosphere. Although nitrogen fixation is exclusively prokaryotic, N 2 -fixing bacteria may be either free-living or living as symbionts with higher plants. For example, Rhizobia are bacteria that fix nitrogen in symbiotic association with soybeans and other legumi- nous plants. Because nitrogen in a metabolically useful form is often the limiting nutrient for plant growth, such symbiotic associations can be an important factor in plant growth and agriculture. Despite the wide diversity of bacteria in which nitrogen fixation takes place, all N 2 -fixing systems are nearly identical and all have four fundamental requirements: (1) the enzyme nitrogenase; (2) a strong reductant, such as reduced ferredoxin; (3) ATP; and (4) O 2 -free conditions. In addition, several modes of regulation act to control nitrogen fixation. The Nitrogenase Complex Is Composed of Two Metalloproteins Two metallopro- teins constitute the nitrogenase complex: the Fe-protein or nitrogenase reductase and the MoFe-protein, which is another name for nitrogenase. Nitrogenase reduc- tase is a 60-kD homodimer possessing a single [4Fe-4S] cluster as a prosthetic group. Nitrogenase reductase is extremely O 2 sensitive. Nitrogenase reductase binds MgATP and hydrolyzes two ATPs per electron transferred during nitrogen fixation. Because reduction of N 2 to 2 NH 4 ϩ ϩ H 2 requires 8 electrons, 16 ATPs are consumed per N 2 reduced. This ATP requirement seems paradoxical because the reaction is thermody- namically favorable: The ⌬Ᏹ o Ј for the reaction (N 2 ϩ 8 e Ϫ ϩ 10 H ϩ → 2 NH 4 ϩ ϩ H 2 ) is –0.314 V. Ferredoxin, the most common e Ϫ donor for nitrogen fixation, has an Ᏹ o Ј that is more negative (see Table 20.1). The solution to the paradox is found in the very strong bonding between the two N atoms in N 2 (Figure 25.4). Sub- stantial energy input is needed to overcome this large activation energy and break the NqN triple bond. In this biological system, the energy is provided by ATP. + e – 8 10 H + + NN 2 NH 4 + H 2 + Energy NN 2 NH 3 Very high energ y of activation –ΔG Reaction coordinate FIGURE 25.4 The triple bond in N 2 must be broken dur- ing nitrogen fixation. 772 Chapter 25 Nitrogen Acquisition and Amino Acid Metabolism Nitrogenase, the MoFe-protein, is a 240-kD ␣ 2 ␤ 2 -type heterotetramer. An ␣␤-dimer serves as the functional unit, and each ␣␤-dimer contains two types of metal centers: an unusual 8Fe-7S center known as the P-cluster (Figure 25.5a) and the novel 7Fe-1Mo-9S cluster known as the FeMo-cofactor (Figure 25.5b). Nitroge- nase under unusual circumstances may contain an ironϺ vanadium cofactor instead of the molybdenum-containing one. Like nitrogenase reductase, nitrogenase is very oxygen labile. The Nitrogenase Reaction In the nitrogenase reaction (Figure 25.6), electrons from reduced ferredoxin pass to nitrogenase reductase, which serves as electron donor to nitrogenase, the enzyme that actually catalyzes N 2 fixation. Electron trans- fer from nitrogenase reductase to nitrogenase takes place through docking of ni- trogenase reductase with an ␣␤-subunit pair of nitrogenase (Figure 25.7). Nitroge- nase reductase transfers e Ϫ to nitrogenase one electron at a time. N 2 is bound within the FeMo-cofactor metal cluster until all electrons and protons are added; no free intermediates, such as HNPNH or H 2 NONH 2 are released. Electron transfer takes place in the following sequence: Fe-protein → P-cluster → FeMo-cofactor → N 2 . ATP hydrolysis is coupled to the transfer of an electron from the Fe-protein to the P-cluster. ATP hydrolysis leads to conformational change in the nitrogenase reduc- (b) Homocitrate Cys␣ 275 Mo Fe S N O His␣ 442 Cys␣ 88 Cys␤ 95 Cys␤ 70 Cys␣ 154 Cys␤ 153 Ser␤ 188 Cys␣ 62 (a) FIGURE 25.5 Structures of the two types of metal clus- ters found in nitrogenase.(a) The P-cluster consists of two Fe 4 S 3 clusters that share an S atom. (b) The FeMo- cofactor contains 1 Mo, 7 Fe, and 9 S atoms.Homocitrate provides two oxo ligands to the Mo atom. (Adapted from Leigh, G. J., 1995.The mechanism of dinitrogen reduction by molybdenum nitrogenases. European Journal of Biochemistry 229:14–20.) e – H 2 Pyruvate h␯ Reduced ferredoxin Nitrogenase reductase (Fe-S) 16 + 16 P i Nitrogenase (Fe-S, FeMoCo) N 2 + 10 H + 2 NH 4 + H 2 8 ATP 16 ADP NADH + FIGURE 25.6 The nitrogenase reaction. Depending on the bacterium, electrons for N 2 reduction may come from light, NADH, hydrogen gas, or pyruvate.The pri- mary e Ϫ donor for the nitrogenase system is reduced ferredoxin.