BI/CH 422/622 OUTLINE: OUTLINE: Protein Degradation (Catabolism) Digestion Amino-Acid Degradation Inside of cells Protein turnover Dealing with the carbon Ubiquitin Fates of the 29 Activation-E1 Seven Families Conjugation-E2 nitrogen atoms in 20 1. ADENQ Ligation-E3 AA: Proteosome 2. RPH 9 ammonia oxidase Amino-Acid Degradation 18 transamination Ammonia 2 urea one-carbon metabolism free transamination-mechanism to know THF Urea Cycle – dealing with the nitrogen SAM 5 Steps Carbamoyl-phosphate synthetase 3. GSC Ornithine transcarbamylase PLP uses Arginino-succinate synthetase Arginino-succinase 4. MT – one carbon metabolism Arginase 5. FY – oxidase vs oxygenase Energetics Urea Bi-cycle 6. KW – Urea Cycle – dealing with the nitrogen 7. BCAA – VIL Feeding the Urea Cycle Glucose-Alanine Cycle Convergence with Fatty acid-odd chain Free Ammonia Overview Glutamine Glutamate dehydrogenase Overall energetics Amino Acid A. Concepts 1. ConvergentDegradation 2. ketogenic/glucogenic 3. Reactions seen before The SEVEN (7) Families B. Transaminase (A,D,E) / Deaminase (Q,N) Family C. Related to biosynthesis (R,P,H; C,G,S; M,T) 1.Glu Family a. Introduce oxidases/oxygenases b. Introduce one-carbon metabolism (1C) 2.Pyruvate Family a. PLP reactions 3. a-Ketobutyric Family (M,T) a. 1-C metabolism D. Dedicated 1. Aromatic Family (F,Y) a. oxidases/oxygenases 2. a-Ketoadipic Family (K,W) 3. Branched-chain Family (V,I,L) E. Convergence with Fatty Acids: propionyl-CoA 29 N 1 Amino Acid Degradation • Intermediates of the central metabolic pathway • Some amino acids result in more than one intermediate. • Ketogenic amino acids can be converted to ketone bodies. Seven to Acetyl-CoA Leu, Ile, Thr, Lys, Phe, Tyr, Trp • Glucogenic amino acids can be converted to glucose. Six to pyruvate1 Ala, Cys, Gly, Ser, Thr, Trp Five to a-ketoglutarate2 Arg, Glu, Gln, His, Pro Four to succinyl-CoA Ile, Met, Thr, Val Two to fumarate3 Phe, Tyr a-ketobutyric Two to oxaloacetate4 Asp, Asn a-keto butyrate family ✓ Branched-chain family 1Pyruvate family ✓ 2Glu family ✓ 3Aromatic family a-keto adipate family 4Trans-/de-aminase family ✓ Amino Acid Degradation F,Y Aromatic Family O2 Phenylalanine hydroxylase NIH-shift H2O Tyrosine aminotransferase 2 4 carbo-cation O2 OH OH – – p-hydroxyphenylpyruvate dioxygenase 6 CO2 NIH-shift 5 4 _ Oxidative 7 ⊕ decarboxylation alpha 3 to a carbonyl 8 2 1 OO Ferryl-peroxyl Homogentisate 1,2-dioxygenase O2 4 5 6 7 8 3 2 1 O 2,4-shift O Maleylacetoacetate isomerase H2O Fumarylacetoacetase Succinate Succinyl-CoA Acetoacetyl-CoA 3-ketoacyl-CoA transferase Acetoacetate 2 Amino Acid Degradation F,Y Aromatic Family O2 Phenylalanine hydroxylase NIH-shift H2O Tyrosine aminotransferase 2 4 carbocation O2 OH OH – – p-hydroxyphenylpyruvate dioxygenase 6 CO2 NIH-shift 5 _ 4 Oxidative 7 ⊕ decarboxylation alpha 3 to a carbonyl 8 2 1 OO Ferryl-peroxyl Homogentisate 1,2-dioxygenase O2 4 5 6 7 8 3 2 1 O 2,4-shift O Maleylacetoacetate isomerase H2O Fumarylacetoacetase Succinate Succinyl-CoA Acetoacetyl-CoA 3-ketoacyl-CoA transferase Acetoacetate Amino Acid Degradation Aromatic Family Genetic Defects in Aromatic Family Degradation Lead to Disease ⊕ ⇀Alkaptonuria • In phenylketonuria (PKU) there is a buildup of Phe and phenylpyruvate – Impairs neurological development leading to severe intellectual deficits – Controlled by limiting dietary intake of Phe • In Alkatonuria there is a buildup of homogentisate, which turns black on oxidation 3 Amino Acid Degradation Aromatic Family Genetic Defects in Aromatic Family Degradation Lead to Disease ⊕ ⇀Alkaptonuria • In phenylketonuria (PKU) there is a buildup of Phe and phenylpyruvate – Impairs neurological development leading to severe intellectual deficits – Controlled by limiting dietary intake of Phe • In Alkatonuria there is a buildup of homogentisate, which turns black on oxidation Amino Acid Degradation: Oxidases Nomenclature Oxidases – use molecular oxygen as an electron acceptor, but no atoms into substrate – e.g., cytochrome oxidase, proline oxidase – usually have water or hydrogen peroxide as product + A(red) + O2 + 4H à A(ox) + 2H2O Oxygenase* – use of molecular oxygen to put into the substrate – dioxygenases – use both atoms; e.g., cysteine dioxygenase A + O2 à AO2 – mono-oxygenases – one atom in substrate, one atom as water AHRH + BH2 + O2 à AOHROH + B + H2O • The enzyme is a cytochrome called P-450 • The co-substrate electrons are from NADPH • There is Cytochrome P-450 reductase to funnel electrons from co-substrate *a.k.a.: hydroxylase, mixed-function oxygenase, “mixed-function oxidase” 4 Amino Acid Degradation: Oxidases Hydroxylases RH + BH2 + O2 à ROH + B + H2O Example of P-450 reactions in adrenal gland: 1) Source of reducing electrons are most often NADPH (the co-substrate) 2) Other co-substrates a) FAD, FeS clusters, a-ketoglutarate (oxidative decarboxylation to succinate) b) Others: tetrahydrobiopterin (Phe hydroxylase) 3) Major class are the P-450 heme hydroxlases a) Expressed in liver and adrenal glands b) Steroids & fatty acids c) Xenobiotics (drugs) i. Specific ii. Non-specific iii. Inducible iv. Drug-drug interactions Amino Acid Degradation F,Y • Intermediates of the central metabolic pathway • Some amino acids result in more than one intermediate. • Ketogenic amino acids can be converted to ketone bodies. Seven to Acetyl-CoA Leu, Ile, Thr, Lys, Phe, Tyr, Trp • Glucogenic amino acids can be converted to glucose. Six to pyruvate1 Ala, Cys, Gly, Ser, Thr, Trp Five to a-ketoglutarate2 Arg, Glu, Gln, His, Pro Four to succinyl-CoA Ile, Met, Thr, Val Two to fumarate3 Phe, Tyr Two to oxaloacetate4 Asp, Asn a-keto butyrate family Branched-chain family 1Pyruvate family 2Glu family 3Aromatic family a-keto adipate family 4Trans-/de-aminase family 5 Amino Acid Degradation K,W a-Ketoadipic Family O Tryptophan 2,3- a-Aminomuconate reductase dioxygenase O N-formyl-L-kynurenine O Kynurenine formamidase a-Amino muconate e- semialdehyde dehydrogenase O Kynurenine 2-Amino muconate e- hydroxylase 2-Amino 3- semialdehyde carboxyl- muconate e- semialdehyde O decarboxylase Kynureninase O O Alanine Muconic acid 3-Hydroxyanthranilic acid 3,4-dioxygenase O Amino Acid Degradation K,W a-Ketoadipic Family O Tryptophan 2,3- a-Aminomuconate reductase dioxygenase O N-formyl-L-kynurenine O Kynurenine formamidase a-Amino muconate e- semialdehyde dehydrogenase O Kynurenine 2-Amino muconate e- hydroxylase 2-Amino 3- semialdehyde carboxyl- muconate e- semialdehyde O decarboxylase Kynureninase O O Alanine Muconic acid 3-Hydroxyanthranilic acid 3,4-dioxygenase O 6 Amino Acid Degradation a-Ketoadipic Family -removal of e-amino -oxidation of e-carbon Lysine:a-ketoglutarate reductase Saccharopine dehydrogenase glutamate 2-aminoadipate aminotransferase a-ketoglutarate PLP a-aminoadipate-d-semialdehyde dehydrogenase Amino Acid Degradation a-Ketoadipic Family -removal of e-amino -oxidation of e-carbon Lysine:a-ketoglutarate reductase Saccharopine dehydrogenase glutamate 2-aminoadipate aminotransferase a-ketoglutarate PLP a-aminoadipate-d-semialdehyde dehydrogenase 7 Amino Acid Degradation K,W • Intermediates of the central metabolic pathway • Some amino acids result in more than one intermediate. • Ketogenic amino acids can be converted to ketone bodies. Seven to Acetyl-CoA Leu, Ile, Thr, Lys, Phe, Tyr, Trp • Glucogenic amino acids can be converted to glucose. Six to pyruvate1 Ala, Cys, Gly, Ser, Thr, Trp Five to a-ketoglutarate2 Arg, Glu, Gln, His, Pro Four to succinyl-CoA Ile, Met, Thr, Val Two to fumarate3 Phe, Tyr Two to oxaloacetate4 Asp, Asn 1.Transaminase/Deaminase Family 2.Glu Family 3.Pyruvate Family 4.a-ketobutyric Family 1 Pyruvate family 5.Aromatic Family 6.a-ketoadipic Family 2Glu family 7.Branched-chain Family 3Aromatic family 4Trans-/de-aminase family Amino Acid Degradation V,I,L • Degradation of branched-chain amino acids does not occur in the liver 7 steps • Leucine, isoleucine, and valine are oxidized for fuel in muscle, adipose tissue, the kidneys, and the brain • Branched-chain amino acids are degraded to succinyl-CoA, an important citric acid cycle CO2 intermediate. 3 steps 5 steps HMG-CoA Ketone Bodies 6 CO2 5 4 3 2 1 b-Hydroxy-methyl-glutAryl-CoA (OMSGAP) 8 Amino Acid Degradation OXIDATIVE DECARBOXYLATION V,I,L Alanine Pyruvate Acetyl-CoA Dehydrogenase (FAD) Cysteine pyruvate dehydrogenase complex Glycine Serine Glutamate Hydratase (H2O) Glutamine a-Ketoglutarate a-ketoglutarate dehydrogenase complex Succinyl-CoA Histidine + Proline Dehydrogenase (NAD ) Arginine Thiolase (CoASH) a-Ketobutyrate Methionine Propionyl-CoA 2 Hydrolase (CoASH) Threonine 2 Carboxylase (biotin) Lysine a-Ketoadipate biotin Glutaconcyl-CoA Crotonyl-CoA Glutaryl-CoAO Tryptophan 2 2 || CO2 2 2Acetyl-CoA | Crotonyl-CoA 2 – | COO – COO CoASH CoASH a-Ketoisovalerate Isobutryl-CoA Methyl OH || O malonyl-CoA a-Keto TPP b-methylvalerate FAD a-Methybutryl-CoA a-methyl- Lipoic acetoacetyl-CoA acid OH || O Acetyl-CoA + Propionyl-CoA CO2 PLP biotin Isovaleryl-CoA a-Keto isocaproate CH2 OH HMG-CoA Amino Acid Degradation OXIDATIVE DECARBOXYLATION V,I,L Alanine Pyruvate Acetyl-CoA Dehydrogenase (FAD) Cysteine pyruvate dehydrogenase complex Glycine Serine Glutamate Hydratase (H2O) Glutamine a-Ketoglutarate a-ketoglutarate dehydrogenase complex Succinyl-CoA Histidine + Proline Dehydrogenase (NAD ) Arginine Thiolase (CoASH) a-Ketobutyrate Methionine Propionyl-CoA 2 Hydrolase (CoASH) Threonine 2 Carboxylase (biotin) Lysine a-Ketoadipate biotin Glutaryl-CoA Glutaconcyl-CoA Crotonyl-CoA Tryptophan 2 2 CO2 2 2Acetyl-CoA | Crotonyl-CoA 2 – | COO – COO CoASH