FCH 532 Lecture 20

1. FCH 532 Lecture 20 Quiz on Wed. Amino acids (25 min) Quiz on Friday Citric Acid Cycle (25 min) Chapter 26: amino acid metabolism New HW posted

2. Amino acid metabolism • Amino acids function as monomers of polypeptides. • Energy metabolites. • Precursors for nitrogen-containing compounds (heme, glutathione, nucleotides, coenzymes) • Amino acids are classified into 2 groups: essential and nonessential • Mammals can synthesize nonessential amino acids from metabolic precursors. • Essential amino acids must be taken in from diet. • Excess dietary amino acids are converted to common metabolic intermediates: pyruvate, OAA, acetyl-CoA, and -ketoglutarate.

3. Breakdown of amino acids • 3 stages • Deamination-the removal of the amino group- conversion to ammonia or the amino group of asp. • Incorporation of ammonia and aspartate nitrogen atoms into urea to be exreted. • Conversion of -keto acids into common metabolic intermediates. Most reactions similar to those covered in other pathways. The first step is deamination of the amino acid.

4. Deamination • Most amino acids use a transamination to deminate the amino acids. • This transfers the amino group of an a-keto acid to make a new amino acids in reactions catalyzed by aminotransferases (aka transaminases). • -ketoglutarate is the predominant amino group acceptor (produces glutamate). Amino acid + -ketoglutarate -ketoacid + glutamate Glutamate’s amino group is then transferred to oxaloacetate to make asp Glutamate + OAA -ketoglutarate + aspartate • Glutamate dehydrogenase (GDH) main catalyst for deamination. Glutamate + NAD(P)+ + H2O -ketoglutarate + NH4+ + NAD(P)H

5. Transamination • Aminotransferase reactions occur in 2 stages: 1. The amino group of an amino acid is transferred to the enzyme: Amino acid + enzyme -keto acid + enzyme-NH2 2. The amino group is transferred to the keto acid acceptor, -ketoglutarate to form glutamate and regenerate the enzyme. -ketoglutarate + enzyme-NH2 enzyme + glutamate • Aminotransferases require the aldehyde-containing coenzyme, pyridoxal-5’-phosphate (PLP) a derivative of pyridoxine (aka vitamin B6). • PLP is attached to the enzyme via a Schiff base linkage by condensation of the aldehyde group to thee -amino group of a Lys within the enzyme. • PLP is converted to pyridoxamine-5’-phosphate (PMP)

6. Figure 26-1ab Forms of pyridoxal-5¢-phosphate.(a) Pyridoxine (vitamin B6) and (b) Pyridoxal-5¢-phosphate (PLP). Page 986

7. Figure 26-1cd Forms of pyridoxal-5¢-phosphate.(c) Pyridoxamine-5¢-phosphate (PMP) and (d) The Schiff base that forms between PLP and an enzyme -amino group. Page 986

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11. Step 1: amino group acts as a nuclophile to attack the enzyme-PLP Schiff base carbon to form an amino acid-PLP-Schiff base (transamination aka trans-Schiffization). This releases the Lys amino group and the Lys can act as a general base catalyst. Page 987

12. Enz-Lys removes the amino acid -carbon H Page 987

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14. Transamination • Can be reversed to convert an -keto acid to an amino acid • PLP functions as an electron sink. • Cleavage of any of the amino acid C atom’s 3 bonds produces a resonance stabilized structure. • PLP can therefore be used in both transamination and decarboxylation reactions. • Most aminotransferases accept only -ketoglutarate or oxaloacetate as the -keto acid substrate in the second stage of the reaction (reverse reaction). • The amino groups of most amino acids are therefore incorporated in the formation of glutamate or aspartate. • Glu and Asp are connected by glutamate-aspartate aminotransferase. Glutamate + oxaloacetate -ketoglutarate + aspartate • Oxidative deamination of glutamate regenerates -ketoglutarate and makes ammonia. • Ammonia and aspartate are the amino donors for urea synthesis.

15. Transamination • Can be reversed to convert an -keto acid to an amino acid • PLP functions as an electron sink. • Cleavage of any of the amino acid C atom’s 3 bonds produces a resonance stabilized structure. • PLP can therefore be used in both transamination and decarboxylation reactions. • Most aminotransferases accept only -ketoglutarate or oxaloacetate as the -keto acid substrate in the second stage of the reaction (reverse reaction). • The amino groups of most amino acids are therefore incorporated in the formation of glutamate or aspartate. • Glu and Asp are connected by glutamate-aspartate aminotransferase. Glutamate + oxaloacetate -ketoglutarate + aspartate • Oxidative deamination of glutamate regenerates -ketoglutarate and makes ammonia. • Ammonia and aspartate are the amino donors for urea synthesis.

16. Glucose-Alanine Cycle • Exception-muscle aminotransferases that accept pyruvate as their -keto acid substrate • Produce alanine to be transported to the liver via the bloodstream. • Once in the liver, Ala is transformed back into pyruvate for use in gluconeogenesis. • Glucose returned to muscle cells to be degraded to pyruvate. During starvation, glucose formed in the liver is used by other tissues and breaks the cycle. Amino groups will be derived from muscle to provide glucose for the other tissues.

17. Figure 26-3 The glucose–alanine cycle. Page 988

18. Oxidative demaniation • Glutamate dehydrogenase (GDH) can use either NAD+ or NADP+ as redox coenzyme. • Allosterically inhibited by GTP and NADH. • Activated by ADP, Leu, and NAD+.

19. Other deamination pathways • Gln made from glutamate and ammonia by glutamine synthestase. N can be transported to the liver from Gln. • Ammonia is released for urea production in the liver mitochondria or for excretion after processing by glutiminase.

20. Other deamination pathways • Gln made from glutamate and ammonia by glutamine synthestase. N can be transported to the liver from Gln. • Ammonia is released for urea production in the liver mitochondria or for excretion after processing by glutiminase.

21. Other deamination pathways • Gln made from glutamate and ammonia by glutamine synthestase. N can be transported to the liver from Gln. • Ammonia is released for urea production in the liver mitochondria or for excretion after processing by glutiminase.

22. Oxidative demaniation • Glutamate dehydrogenase (GDH) can use either NAD+ or NADP+ as redox coenzyme. • Allosterically inhibited by GTP and NADH. • Activated by ADP, Leu, and NAD+.

23. Glu Figure 26-5a X-Ray structures of glutamate dehydrogenase (GDH). (a) Bovine GDH in complex with glutamate, NADH, and GTP. NADH Page 990 GTP NADH bound at ADP effector site

24. Antenna domain Figure 26-5b X-Ray structures of glutamate dehydrogenase (GDH). (b) One subunit of the bovine GDH–glutamate–NADH–GTP complex. Pivot helix NADH GTP Coenzyme binding domain Page 990 Substrate binding domain NADH Glu

25. Figure 26-5c X-Ray structures of glutamate dehydrogenase (GDH). (c) One subunit of human apoGDH with the protein colored as and viewed similarly to Part b. Binding rotates about pivot helix causing cleft to close Page 990

26. Figure 26-6 Inhibition of human glutamate dehydrogenase (GDH) by GTP.(50% inhibition at midpoint) Page 990

27. Other deamination pathways • Nonspecific amino acid oxidases - L-amino acid oxidase and D-amino acid oxidase. • Have FAD as redox coenzyme. Amino acid + FAD + H2O -keto acid + NH3 + FADH2 FADH2 + O2 FAD + H2O2

28. Urea Cycle • Excess nitrogen is excreted after the metabolic breakdown of amino acids in one of three forms: • Aquatic animals are ammonotelic (release NH3 directly). • If water is less plentiful, NH3 is converted to less toxic products, urea and uric acid. • Terrestrial vertebrates are ureotelic (excrete urea) • Birds and reptiles are uricotelic (excrete uric acid) • Urea is made by enzymes urea cycle in the liver. • The overall reaction is: NH3+ NH3 + HCO3-+ -OOC-CH2-CH-COO- Asp 3ATP 2ADP + 2Pi + AMP + PPi O NH2-C-NH2+ -OOC-CH=CH-COO- Fumarate Urea

29. Urea Cycle • 2 urea nitrogen atoms come from ammonia and aspartate. • Carbon atom comes from bicarbonate. • 5 enzymatic reactions used, 2 in the mitochondria and 3 in the cytosol. NH3+ NH3 + HCO3-+ -OOC-CH2-CH-COO- Asp 3ATP 2ADP + 2Pi + AMP + PPi O NH2-C-NH2+ -OOC-CH=CH-COO- Fumarate Urea

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31. O 2ATP + NH3 + HCO3- NH2-C-OPO3- + 2ADP + 2Pi Carbamoyl phosphate Carbamoyl phosphate synthetase • Carbamoyl phosphate synthetase (CPS) catalyzes the condensation and activation NH3 and HCO3- to form carbomyl phosphate (first nitrogen containing substrate). • Uses 2 ATPs. • Eukaryotes have 2 types of CPS enzymes • Mitochondrial CPSI uses NH3 as its nitrogen donor and participates in urea biosynthesis. • Cytosolic CPSII uses glutamine as its nitrogen donor and is involved in pyrimidine biosynthesis.

32. Figure 26-8 The mechanism of action of CPS I. • CPSI reaction has 3 steps • Activation of HCO3- by ATP to form carboxyphosphate and ADP. • Nucelophilic attack of NH3 on carboxyphosphate, displacing the phsophate to form carbamate and Pi. • Phosphorylation of carbamate by the second ATP to form carbamoyl phosphate and ADP The reaction is irreversible. Allosterically activated by N-acetylglutamate. Page 993

33. Figure 26-9 X-Ray structure of E. coli carbamoyl phosphate synthetase (CPS). • E. coli has only one CPS (homology to CPS I and CPS II) • Heterodimer (inactive). • Allosterically activated by ornithine (heterotetramer of (4). • Small subunit hydrolyzes Gln and delivers NH3 to large subunit. • Channels intermediate of two reactions from one active site to the other. Page 993

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35. Ornithine transcarbomylase • Transfers the carbomoyl group of carbomyl phosphate to ornithine to make citrulline • Reaction occurs in mitochondrion. • Ornithine produced in the cytosol enters via a specific transport system. • Citrulline is exported from the mitochondria.

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37. Arginocuccinate Synthetase • 2nd N in urea is incorporated in the 3rd reaction of the urea cycle. • Condensation reaction with citrulline’s ureido group with an Asp amino group catalyzed by arginosuccinate synthetase. • Ureido oxygen is activated as a leaving group through the formation of a citrulyl-AMP intermediate. • This is displaced by the Asp amino group to form arginosuccinate.

38. Figure 26-10 The mechanism of action of argininosuccinate synthetase. Page 994

39. Arigininosuccinase and Arginase • Argininosuccinse catalyzes the elimination of Arg from the the Asp carbon skeleton to form fumurate. • Arginine is the immediate precursor to urea. • Fumurate is converted by fumarase and malate dehydrogenase to to form OAA for gluconeogenesis. • Arginase catalyzes the fifth and final reaction of the urea cycle. • Arginine is hydrolyzed to form urea and regenerate ornithine. • Ornithine is returned to the mitochondria.

40. Carbamoyl phosphate synthetase (CPS) Ornithine transcarbamoylase Argininosuccinate synthetase Arginosuccinase Arginase Page 992

41. Regulation of the urea cycle • Carbamoyl phosphate synthetase I is allosterically activated by N-acetylglutamate. • N-acetylglutamate is synthesized from glutamate and acetyl-CoA by N-acetylglutamate synthase, it is hydrolyzed by a specific hydrolase. • Rate of urea production is dependent on [N-acetylglutamate]. • When aa breakdown rates increase, excess nitrogen must be excreted. This results in increase in Glu through transamination reactions. • Excess Glu causes an increase in N-acetylglutamate which stimulates CPS I causing increases in urea cycle.

42. Metabolic breakdown of amino acids • Degradation of amino acids converts the to TCA cycle intermediates or precursors to be metabolized to CO2, H2O, or for use in gluconeogenesis. • Aminoacids are glucogenic, ketogenic or both. • Glucogenic amino acids-carbon skeletons are broken down to pyruvate, -ketoglutarate, succinyl-CoA, fumarate, or oxaloacetate (glucose precursors). • Ketogenic amino acids, are broken down to acetyl-CoA or acetoacetate and therefore can be converted to fatty acids or ketone bodies.