Molecular Biochemistry II Amino Acid Catabolism: N Copyright © 1999-2008 by Joyce J.
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Molecular Biochemistry II Amino Acid Catabolism: N Copyright © 1999-2008 by Joyce J. Diwan. All rights reserved. H R1 C COO NH3 Transaminases (aminotransferases) catalyze the reversible reaction at right. - + R2 + C COO - O Transaminase H R1 C COO O - + R2 C COO NH3 - + 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 a-keto acid acceptors. COO COO COO CH2 COO CH2 CH2 CH2 CH2 CH2 HC NH3+ COO + C O COO C O + COO HC NH3+ COO aspartate a-ketoglutarate oxaloacetate glutamate Aminotransferase (Transaminase) Example of a Transaminase reaction: Aspartate donates its amino group, becoming the a-keto acid oxaloacetate. a-Ketoglutarate accepts the amino group, becoming the amino acid glutamate. CH3 HC COO COO CH2 CH2 CH2 NH3+ COO alanine + C CH3 O COO a-ketoglutarate C CH2 O COO + HC NH3+ COO pyruvate glutamate Aminotransferase (Transaminase) In another example, alanine becomes pyruvate as the amino group is transferred to a-ketoglutarate. Transaminases equilibrate amino groups among available a-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). Essential amino acids must be consumed in the diet. Mammalian cells lack enzymes to synthesize their carbon skeletons (a-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.) H O O P O O C H2 C OH O N H CH3 pyridoxal phosphate (PLP) The prosthetic group of Transaminase is pyridoxal phosphate (PLP), a derivative of vitamin B6. R H C COO Enz (CH2)4 NH2 Amino acid N+ HC O O H2 C P O H O O N H CH3 Enzyme (Lys)-PLP Schiff base In the resting state, the aldehyde group of pyridoxal phosphate is in a Schiff base linkage to the e-amino group of an enzyme lysine side-chain. EnzLysNH2 R H C COO N+ The a-amino group of a substrate amino acid displaces the enzyme lysine, to form a Schiff base linkage to PLP. HC O O H2 C P O H O O N H CH3 Amino acid-PLP Shiff base (aldimine) The active site lysine extracts H+, promoting tautomerization, followed by reprotonation & hydrolysis. O EnzLysNH2 H2 C P O What was an amino acid leaves as an a-keto acid. CH2 O O NH2 R C COO a-keto acid OH O N CH3 H Pyridoxamine phosphate (PMP) The amino group remains on what is now pyridoxamine phosphate (PMP). A different a-keto acid reacts with PMP and the process reverses, to complete the reaction. EnzLysNH2 R H C COO N+ HC O O H2 C P O H O O N H CH3 Amino acid-PLP Shiff base (aldimine) 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. 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 a-ketoglutarate, at the active site. Students should then show and explain the structure displayed by them to the neighboring student or student group. In addition to equilibrating amino groups among available a-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. OOC Glutamate Dehydrogenase catalyzes a major reaction that effects net removal of N from the amino acid pool. H2 C H2 C glutamate NH3+ C H H 2O COO NAD(P)+ NAD(P)H O H2 H2 OOC C C a-ketoglutarate C COO + NH4+ Glutamate Dehydrogenase It is one of the few enzymes that can use NAD+ or NADP+ as e acceptor. Oxidation at the a-carbon is followed by hydrolysis, releasing NH4+. Amino acid a-ketoglutarate a-keto acid glutamate Transaminase + NADH + NH4 + NAD + H2O Glutamate Dehydrogenase Summarized above: The role of transaminases in funneling amino N to glutamate, which is deaminated via Glutamate Dehydrogenase, producing NH4+. + H2O NH4 H2O HO CH2 H C COO NH3+ serine H2C C COO O H3C C COO NH3+ aminoacrylate pyruvate Serine Dehydratase Some other pathways for deamination of amino acids: 1. Serine Dehydratase catalyzes: serine pyruvate + NH4+ 2. Peroxisomal L- and D-amino acid oxidases catalyze: amino acid + FAD + H2O a-keto acid + NH4+ + FADH2 FADH2 + O2 FAD + H2O2 Catalase catalyzes: 2H2O2 2 H2O + O2 O H2N C NH2 urea 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 NH3 (produced mainly via Glutamate Dehydrogenase) and as the amino N of aspartate. The NH3 and HCO3 (carbonyl C) that will be part of urea are incorporated first into carbamoyl phosphate. HCO3 Carbamoyl Phosphate Synthase (Type I) catalyzes a 3-step reaction, with carbonyl phosphate and carbamate intermediates. ATP ADP O HO carbonyl phosphate NH3 Pi O Ammonia is the N input. The reaction, which involves cleavage of 2 ~P bonds of ATP, is essentially irreversible. OPO 32 C H2N C O carbamate ATP ADP O H2N C OPO 32 carbamoyl phosphate HCO3 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. ATP ADP O HO OPO 32 C carbonyl phosphate NH3 Pi O H2N C O carbamate ATP ADP O H2N C OPO 32 carbamoyl phosphate HCO3 + NH3 + 2 ATP O H2N C Carbamoyl Phosphate Synthase OPO 32 + 2 ADP + Pi carbamoyl phosphate Carbamoyl Phosphate Synthase is the committed step of the Urea Cycle, and is subject to regulation. glutamate (Glu) N-acetylglutamate H H3N+ C O COO H 3C C H N H C COO CH2 CH2 CH2 CH2 COO COO 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. O NH3+ Urea Cycle C CH2 NH3+ NH ornithine 4 O H2O citrulline CH2 Pi CH2 COO C urea NH2 3 COO CH2 HC CH2 HC HC H N COO NH arginine CH NH3+ COO NH2 COO aspartate CH2 C CH2 HC NH2+ H2N CH2 2 ATP AMP + PPi COO H2N COO NH3+ HC Urea Cycle COO NH2 CH2 1 CH2 HC OPO32 carbamoyl phosphate CH2 Enzymes in mitochondria: 1. Ornithine Transcarbamylase Enzymes in cytosol: 2. ArgininoSuccinate Synthase 3. Argininosuccinase 4. Arginase. H2N C O COO fumarate C NH2+ NH CH2 CH2 argininosuccinate CH2 HC NH3+ COO cytosol mitochondrial matrix carbamoyl phosphate Pi ornithine citrulline ornithine urea arginine citrulline aspartate argininosuccinate fumarate 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. cytosol mitochondrial matrix carbamoyl phosphate Pi ornithine citrulline ornithine urea arginine citrulline aspartate argininosuccinate fumarate A complete Krebs Cycle functions only within mitochondria. But cytosolic isozymes of some Krebs Cycle enzymes are involved in regenerating aspartate from fumarate. COO COO COO CH2 COO CH2 CH2 CH2 CH2 CH2 HC NH3+ COO + C O COO C O COO + HC NH3+ COO aspartate a-ketoglutarate oxaloacetate glutamate Aminotransferase (Transaminase) 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. 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. Postulated mechanisms for toxicity of high [ammonia]: 1. High [NH3] would drive Glutamine Synthase: glutamate + ATP + NH3 glutamine + ADP + Pi 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)+ a-ketoglutarate + NAD(P)H + NH4+ The resulting depletion of a-ketoglutarate, an essential Krebs Cycle intermediate, could impair energy metabolism in the brain. 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 a-keto acid analogs of essential amino acids. Liver transplantation has also been used, since liver is the organ that carries out Urea Cycle. cytosol The complete Urea Cycle is significantly only in liver. mitochondrial matrix carbamoyl phosphate Pi ornithine citrulline However some ornithine citrulline urea aspartate enzymes of the arginine argininosuccinate pathway are in fumarate other cells and 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. arginine (Arg) H H3N+ C COO CH2 CH3 CH2 H2N CH2 N CH2 NH2+ NH C C O C O creatine NH2 NH2 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. NH2 C + NH2 NH C NADPH NADP+ CH2 O2 CH C OH CH2 O2 arginine + H3 N CH + NO CH2 H2O CH2 CH2 COO O NH 1/2 NADPH 1/2 NADP+ CH2 H2O CH2 H3N NH N CH2 CH2 + NH2 NH2 COO hydroxyarginine + H3N CH COO citrulline Nitric Oxide Synthase Synthesis of the radical species nitric oxide (·NO) from arginine is catalyzed Nitric Oxide Synthase, a distant relative of cytochrome P450. Different isoforms of Nitric Oxide Synthase (e.g., eNOS expressed in endothelial cells and nNOS in neuronal cells) are subject to differing regulation. ·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 (O2·) to form the strong oxidant peroxynitrite (ONOO). + + H3N CH2 CH2 CH2 CH2 H3N CH2 CH2 NH CH2 NH3+ CH2 putrescine CH2 CH2 CH2 NH3+ spermidine Polyamines include putrescine, spermidine, spermine. Ornithine is a major precursor for synthesis of polyamines. Conversion of ornithine to putrescine is catalyzed by Ornithine Decarboxylase. H H3N+ C COO CH2 CH2 CH2 NH3 ornithine + + H3N CH2 CH2 CH2 CH2 H3N CH2 CH2 NH CH2 NH3+ CH2 putrescine CH2 CH2 CH2 NH3+ spermidine The cationic polyamines have diverse roles in cell growth & proliferation. Disruption of polyamine synthesis or metabolism leads to disease in animals & humans. H H3N+ There is no tRNA for citrulline & this amino acid is not incorporated translationally into proteins. C H COO H3N+ C CH2 CH2 CH2 CH2 CH2 CH2 NH NH COO C NH2 NH2 arginine C NH2 O citrulline However, Ca++-activated Peptidylarginine Deiminases convert arginine residues within proteins to citrulline as a post-translational modification. H H3N+ Substitution of citrulline, which lacks arginine's positive charge, may alter structure & properties such as binding affinities of a protein. C H COO H3N+ C CH2 CH2 CH2 CH2 CH2 CH2 NH NH COO C NH2 C NH2 NH2 O E.g., citrullination of certain arginine citrulline proteins, including keratin intermediate filament proteins, 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.