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Amino Acid Catabolism: N Copyright © 1999-2008 by Joyce J. Diwan. All rights reserved. Molecular Biochemistry II.

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Presentation on theme: "Amino Acid Catabolism: N Copyright © 1999-2008 by Joyce J. Diwan. All rights reserved. Molecular Biochemistry II."— Presentation transcript:

1 Amino Acid Catabolism: N Copyright © 1999-2008 by Joyce J. Diwan. All rights reserved. Molecular Biochemistry II

2 There are multiple transaminase enzymes which vary in substrate specificity. Some show preference for particular amino acids or classes of amino acids as amino group donors, and/or for particular  -keto acid acceptors. Transaminases (aminotransferases) catalyze the reversible reaction at right.

3 Example of a Transaminase reaction:  Aspartate donates its amino group, becoming the  -keto acid oxaloacetate.   -Ketoglutarate accepts the amino group, becoming the amino acid glutamate.

4 In another example, alanine becomes pyruvate as the amino group is transferred to  -ketoglutarate.

5 Transaminases equilibrate amino groups among available  -keto acids. This permits synthesis of non-essential amino acids, using amino groups from other amino acids & carbon skeletons synthesized in a cell. Thus a balance of different amino acids is maintained, as proteins of varied amino acid contents are synthesized. Although the amino N of one amino acid can be used to synthesize another amino acid, N must be obtained in the diet as amino acids (proteins).

6 Essential amino acids must be consumed in the diet. Mammalian cells lack enzymes to synthesize their carbon skeletons (  -keto acids). These include: Isoleucine, leucine, & valine Lysine Threonine Tryptophan Phenylalanine (Tyr can be made from Phe.) Methionine (Cys can be made from Met.) Histidine (Essential for infants.)

7 The prosthetic group of Transaminase is pyridoxal phosphate (PLP), a derivative of vitamin B 6.

8 In the resting state, the aldehyde group of pyridoxal phosphate is in a Schiff base linkage to the  -amino group of an enzyme lysine side-chain.

9 The active site lysine extracts H +, promoting tautomerization, followed by reprotonation & hydrolysis. The  -amino group of a substrate amino acid displaces the enzyme lysine, to form a Schiff base linkage to PLP.

10 The amino group remains on what is now pyridoxamine phosphate (PMP). A different  -keto acid reacts with PMP and the process reverses, to complete the reaction. What was an amino acid leaves as an  -keto acid.

11 Several other enzymes that catalyze metabolism or synthesis of amino acids also utilize PLP as prosthetic group, and have mechanisms involving a Schiff base linkage of the amino group to PLP.

12 Chime Exercise Two neighboring students or student groups should team up, each displaying one of the following:  Transaminase with PLP in Schiff base linkage to the active site lysine residue.  Transaminase in the PMP form, with glutarate, an analog of  -ketoglutarate, at the active site. Students should then show and explain the structure displayed by them to the neighboring student or student group.

13 In addition to equilibrating amino groups among available  -keto acids, transaminases funnel amino groups from excess dietary amino acids to those amino acids (e.g., glutamate) that can be deaminated. Carbon skeletons of deaminated amino acids can be catabolized for energy, or used to synthesize glucose or fatty acids for energy storage. Only a few amino acids are deaminated directly.

14 It is one of the few enzymes that can use NAD + or NADP + as e  acceptor. Oxidation at the  -carbon is followed by hydrolysis, releasing NH 4 +. Glutamate Dehydrogenase catalyzes a major reaction that effects net removal of N from the amino acid pool.

15 Summarized above: The role of transaminases in funneling amino N to glutamate, which is deaminated via Glutamate Dehydrogenase, producing NH 4 +.

16 Some other pathways for deamination of amino acids: 1. Serine Dehydratase catalyzes: serine  pyruvate + NH 4 + 2. Peroxisomal L- and D-amino acid oxidases catalyze: amino acid + FAD + H 2 O   -keto acid + NH 4 + + FADH 2 FADH 2 + O 2  FAD + H 2 O 2 Catalase catalyzes: 2 H 2 O 2  2 H 2 O + O 2

17 Most terrestrial land animals convert excess nitrogen to urea, prior to excreting it. Urea is less toxic than ammonia. The Urea Cycle occurs mainly in liver. The 2 nitrogen atoms of urea enter the Urea Cycle as NH 3 (produced mainly via Glutamate Dehydrogenase) and as the amino N of aspartate. The NH 3 and HCO 3  (carbonyl C) that will be part of urea are incorporated first into carbamoyl phosphate.

18 Carbamoyl Phosphate Synthase (Type I) catalyzes a 3-step reaction, with carbonyl phosphate and carbamate intermediates. Ammonia is the N input. The reaction, which involves cleavage of 2 ~P bonds of ATP, is essentially irreversible.

19 Alternate forms of Carbamoyl Phosphate Synthase (Types II & III) initially generate ammonia by hydrolysis of glutamine. The type II enzyme includes a long internal tunnel through which ammonia & reaction intermediates such as carbamate pass from one active site to another.

20 Carbamoyl Phosphate Synthase is the committed step of the Urea Cycle, and is subject to regulation.

21 Carbamoyl Phosphate Synthase has an absolute requirement for an allosteric activator N-acetylglutamate. This derivative of glutamate is synthesized from acetyl-CoA & glutamate when cellular [glutamate] is high, signaling an excess of free amino acids due to protein breakdown or dietary intake.

22 Urea Cycle Enzymes in mitochondria: 1. Ornithine Trans- carbamylase Enzymes in cytosol: 2. Arginino- Succinate Synthase 3. Arginino- succinase 4. Arginase.

23 For each cycle, citrulline must leave the mitochondria, and ornithine must enter the mitochondrial matrix. An ornithine/citrulline transporter in the inner mitochondrial membrane facilitates transmembrane fluxes of citrulline & ornithine.

24 A complete Krebs Cycle functions only within mitochondria. But cytosolic isozymes of some Krebs Cycle enzymes are involved in regenerating aspartate from fumarate.

25 Fumarate is converted to oxaloacetate via Krebs Cycle enzymes Fumarase & Malate Dehydrogenase. Oxaloacetate is converted to aspartate via transamination (e.g., from glutamate). Aspartate then reenters Urea Cycle, carrying an amino group derived from another amino acid.

26 Hereditary deficiency of any of the Urea Cycle enzymes leads to hyperammonemia - elevated [ammonia] in blood. Total lack of any Urea Cycle enzyme is lethal. Elevated ammonia is toxic, especially to the brain. If not treated immediately after birth, severe mental retardation results.

27 Postulated mechanisms for toxicity of high [ammonia]: 1. High [NH 3 ] would drive Glutamine Synthase: glutamate + ATP + NH 3  glutamine + ADP + P i This would deplete glutamate – a neurotransmitter & precursor for synthesis of the neurotransmitter GABA. 2. Depletion of glutamate & high ammonia level would drive Glutamate Dehydrogenase reaction to reverse: glutamate + NAD(P) +   -ketoglutarate + NAD(P)H + NH 4 + The resulting depletion of  -ketoglutarate, an essential Krebs Cycle intermediate, could impair energy metabolism in the brain.

28 Treatment of deficiency of Urea Cycle enzymes (depends on which enzyme is deficient):  limiting protein intake to the amount barely adequate to supply amino acids for growth, while adding to the diet the  -keto acid analogs of essential amino acids.  Liver transplantation has also been used, since liver is the organ that carries out Urea Cycle.

29 tissues where they generate arginine & ornithine, which are precursors for other important molecules. E.g., Argininosuccinate Synthase, which catalyzes synthesis of the precursor to arginine, is in most tissues. Mitochondrial Arginase II, distinct from the cytosolic Urea Cycle Arginase, cleaves arginine to yield ornithine. The complete Urea Cycle is significantly only in liver. However some enzymes of the pathway are in other cells and

30 The amino acid arginine, in addition to being a constituent of proteins and an intermediate of the Urea Cycle, is precursor for synthesis of creatine & the signal molecule nitric oxide.

31 Synthesis of the radical species nitric oxide (·NO) from arginine is catalyzed Nitric Oxide Synthase, a distant relative of cytochrome P 450. Different isoforms of Nitric Oxide Synthase (e.g., eNOS expressed in endothelial cells and nNOS in neuronal cells) are subject to differing regulation.

32  ·NO is a short-lived signal molecule with diverse roles in different cell types, including regulation of smooth muscle contraction, gene transcription, metabolism, and neurotransmission. Many of the regulatory effects of ·NO arise from its activation of a soluble cytosolic Guanylate Cyclase enzyme that catalyzes synthesis of cyclic-GMP (analogous in structure to cyclic-AMP).  Cytotoxic effects of ·NO observed under some conditions are attributed to its non-enzymatic reaction with superoxide (O 2 ·  ) to form the strong oxidant peroxynitrite (ONOO  ).

33  Polyamines include putrescine, spermidine, spermine.  Ornithine is a major precursor for synthesis of polyamines. Conversion of ornithine to putrescine is catalyzed by Ornithine Decarboxylase.

34  The cationic polyamines have diverse roles in cell growth & proliferation. Disruption of polyamine synthesis or metabolism leads to disease in animals & humans.

35 However, Ca ++ -activated Peptidylarginine Deiminases convert arginine residues within proteins to citrulline as a post-translational modification. There is no tRNA for citrulline & this amino acid is not incorporated translationally into proteins.

36 is essential to terminal differentiation of skin cells. Excessive protein citrullination, with production of antibodies against citrullinated proteins, is found to be a factor in the autoimmune diseases such as rheumatoid arthritis and multiple sclerosis. Substitution of citrulline, which lacks arginine's positive charge, may alter structure & properties such as binding affinities of a protein. E.g., citrullination of certain proteins, including keratin intermediate filament proteins,

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