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Amino acid metabolism · Nitrogen balance Dietary protein amino acid pool protein synthesis catabolism, biosynthesis normal N balance: N ingested = N excreted negative N balance: N ingested < N excreted positive N balance: N ingested > N excreted N excretion (NH4+. urea) Requirement for essential amino acids Amino acid catabolism · accounts for ~ 10% of energy requirement of adults · When: • · excess protein in diet (amino acids are not stored) • · protein degradation exceeds demand for new protein • · starvation when carbohydrates are not available · (protein storing seeds such as beans, peas, etc.) · Glucogenic vs ketogenic amino acids · ketogenic: yield AcCoA or AcAc as end products of catabolism - leu, lys · glucogenic: are degraded to pyruvate or a member of the TCA cycle (succinylCoA, OAA, a-ketoglutarate, fumarate). In absence of sugars, glucogenic amino acids permit continued oxidation of fatty acids by maintaining TCA cycle intermediates. Also source of carbons for gluconeogenesis in liver - ile, phe, tyr, trp · glucogenic and ketogenic: yield both ketogenic and glucogenic products. - all others N catabolism General strategy: 1 removal of N from amino acid by transamination (generally first or second step of amino acid catabolic pathways) and collection of N in glutamic acid 2 deamination of glutamic acid with release of NH4+ -glutamate dehydrogenase 3. Collection of N in glutamine or alanine for delivery to liver 4 removal of NH4+ by : i. secretion; or ii. conversion to urea or other less toxic form. Vitamine B6 family Pyridoxine Pyridoxal Pyridoxamine to e-amino of lysine Pyridoxal phosphate See Horton: page 212 section 7.7 pyridoxal phosphate 1. Transamination reaction see text p 537 and fig 17.7. Lys-protein NH R1 H- C-NH3+ COO- + a-aminoacid-1 Schiff base with enzyme R1 H-C-COONH Lys-protein Schiff base with substrate R1 H-C-COONH Lys-protein Schiff base with substrate NH2 Lys-protein R1 + H- C- O COO- a-ketoacid-1 Pyradoxamine phosphate NH2 Lys-protein R2 + H- C- O COO- a-ketoacid-2 R2 H-C-COONH Lys-protein R2 H-C-COO- NH Lys-protein Lys-protein NH R2 H- C-NH3+ COO- a-amino acid-2 + Net reaction: a-amino acid-1 + a-ketoacid-2 PLP a-amino acid-2 + a-ketoacid-1 e.g. alanine + a-ketoglutarate pyruvate + glutamate N catabolism General strategy: 1 removal of N from amino acid by transamination (generally first or second step of amino acid catabolic pathways) and collection of N in glutamic acid 2 deamination of glutamic acid with release of NH4+ -glutamate dehydrogenase 3. Collection of N in glutamine or alanine for delivery to liver 4 removal of NH4+ by : i. secretion; or ii. conversion to urea or other less toxic form. 2. glutamate dehydrogenase (see p 533 for reaction) • - release or capture of NH4+ · - located in mitochondria · - operates near equilibrium NAD NADH a-ketoglutarate + NH4+ glutamate + H2O NADP amino acid + a-ketoglutar glutamate + NAD + H2O amino acid + NAD + H2O NADPH a-keto acid + glutamate a-ketoglutar +NADH + H+ + NH4+ a-keto acid +NADH + H+ + NH4+ 3. transport of N to the liver - glutamine synthetase - glutaminase - alanine/glucose cycle 1. Glutamine synthetase ATP glutamate + NH4 2. Glutaminase glutamine ADP + Pi + glutamine glutamate + NH4+ Note: glutamate can be used for glucose synthesis. How? 3. Formation of alanine by transamination: alanine/glucose cycle Alanine-glucose cycle Muscle glucose 2 pyruvate 2 a-aa 2 a-ka 2 alanine 2 alanine glucose Liver glucose 2 NH4+ 2 pyruvate 2 Glu 2 a-kG 2 alanine MUSCLE energy protein Glu’NH2 NH4+ a-ka Pyr Glu Glucose a-aa a-KG Ala Pyr Ala a-KG Glucose Glu’NH2 Glu CO2 NH4 LIVER Glucose CO2 H2O Urea 2NH4+ 2NH4+ 2a-KG + 2Glu 4CO2 2Glu’NH2 Urea KIDNEY H2CO3 HCO3 + H+ Urea cycle Where: Liver: mito/cyto Why: disposal of N Immediate source of N: glutamate dehydrogenase glutaminase Fate of urea: liver kidney How much: ~ 30g urea / day urine Reactions of urea cycle 1. Carbamyl phosphate synthetase I (mito) NH4+ + HCO3- + 2 ATP O H2N-C-OPO3-2 + Pi + 2 ADP carbamyl phosphate • committed step • by N’Ac glutamate 2. Ornithine transcarbamylase (mito) NH2 CH2 CH2 CH2 CH COONH3+ ornithine + NH 2 C O OPO 3-2 carbamyl phosphate Pi NH2 C O HN CH2 CH2 CH2 CH COONH3+ citrulline 3. Arginosuccinate synthetase (cyto) NH2 C O HN CH2 CH2 CH2 CH COO- COO- H2N COO- + CH2 CH NH3+ COO- NH3+ ATP AMP + PPi C NH HN CH2 CH2 CH2 CH COO- CH CH2 COO- NH3+ arginosuccinate 4. Arginosuccinate lyase (cyto) H2N C NH HN CH2 CH2 CH2 CH COONH3+ COOCH CH2 COO- H2 N C NH2 HN CH2 CH2 CH2 CH COONH3+ arginine COO- + CH CH COO- fumarate 5. Arginase (cyto) H2 N C NH2 HN CH2 CH2 CH2 CH COONH3+ NH2 CH2 CH2 + CH2 CH COONH3+ ornithine NH2 C NH2 urea O NH 2 C O OPO 3-2 NH3+ 2ATP 2ADP +Pi NH 2 CH 2 CH 2 CH 2 CH COONH 3+ + ornithine NH 2 C O HN CH 2 CH 2 CH 2 CH COO- HCO3 NADH + H+ NAD NH 3+ MITO C CYTO ornithine NH 2 aKG citrulline asparate glutamate asparate glutamate O ATP NH 2 AMP + PPi H2N H2 N C NH 2 HN CH 2 CH 2 CH 2 CH COO- C NH HN CH2 CH2 CH2 CH COONH3+ NH 3+ fumarate COOCH CH 2 COO- See fig 17.26 Interorgan relationships in N metabolism Glu’NH2 Several steps Epithelial cells of intestine cittruline Glu’NH2 Liver Kidney cittruline Arg Urea Urea cycle Ornithine 2 steps glutamate Arginine Arginine Several steps creatine To urine Muscle creatine creatinine P-creatine Several steps Adapted from Devlin, Biochemistry with Clinical Corrleation