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 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
Isovaleryl-CoA a-Keto isocaproate CH2 OH
HMG-CoA
9 Amino Acid Degradation V,I,L
• 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
1Pyruvate family 2Glu family 3Aromatic family 4Trans-/de-aminase family
Amino Acid Degradation Meets Fatty Acid Degradation Oxidation of Propionyl-CoA Threonine thymidines Valine
Odd-Chain Fatty Acids
L–
( R ) ( R )
10 Amino Acid Degradation Meets Fatty Acid Degradation Oxidation of Propionyl-CoA Threonine thymidines Valine
Odd-Chain Fatty Acids
L–
( R ) ( R )
Amino Acid Degradation Meets Fatty Acid Degradation
Only a handful of enzymes require Coenzyme B12 Diet Vitamin B12 (cobalamin) (OH-Cbl)
Saliva
IF-1 Plasma (OH-Cbl)
Stomach
TC-1 (OH-Cbl) H2O (Co3+)
11 Amino Acid Degradation Meets Fatty Acid Degradation Complex Cobalt-Containing Compound: Coenzyme B12
Nobel Prize
1957 1964
Dr. Kornberg: Lecture 16 02.27.17 (28:19- 29:33) VitB-12 1.3 min
Amino Acid Degradation Meets Fatty Acid Degradation
12 Amino Acid Degradation: Overview
Glutamine Histidine Proline Arginine
4 Steps 2 Steps 2 Steps
THF Glu-semialdehyde
5 Steps Leucine HMG-CoA Isoleucine Valine 3 Steps SAM Methionine 2 Steps 2 Steps 2 Steps 4 Steps cycle 5 Steps 3 Steps Propionyl-CoA a-ketobutyric Threonine
5 Steps 5/6 Steps glutaryl-CoA Cysteine 4 Steps Lysine a-ketoadipic 3 Steps 1.Transaminase/Deaminase Family 2.Glu Family 3.Pyruvate Family 5 Steps 4.a-ketobutyric Family 3 Steps 5.Aromatic Family Tryptophan Alanine 6.a-ketoadipic Family 7.Branched-chain Family THF Serine cycle
Glycine
CO2 + NH4+ Oxidative decarboxylation by a complex yielding NADH Asparagine
Amino Acid Degradation: Overview
TABLE 18-2 Some Human Genetic Disorders Affecting Amino Acid Catabolism
Approximate incidence (per Medical condition 100,000 births) Defective process Defective enzyme Symptoms and effects Albinism <3 Melanin synthesis from Tyrosine 3- Lack of pigmentation; tyrosine monooxygenase white hair, pink skin (tyrosinase) Alkaptonuria <0.4 Tyrosine degradation Homogentisate 1,2- Dark pigment in urine; dioxygenase late-developing arthritis Argininemia <0.5 Urea synthesis Arginase Mental retardation
Argininosuccinic acidemia <1.5 Urea synthesis Argininosuccinase Vomiting; convulsions
Carbamoyl phosphate synthetase I <0.5 Urea synthesis Carbamoyl phosphate Lethargy; convulsions; deficiency synthetase I early death Homocystinuria <0.5 Methionine degradation Cystathionine β- Faulty bone synthase development; mental retardation Maple syrup urine disease <0.4 Isoleucine, leucine, and Branched-chain α-keto Vomiting; convulsions; (branchedchain ketoaciduria) valine degradation acid dehydrogenase mental retardation; complex early death Methylmalonic acidemia <0.5 Conversion of Methylmalonyl-CoA Vomiting; convulsions; propionyl-CoA to mutase mental retardation; succinyl-CoA early death Phenylketonuria <8 Conversion of Phenylalanine Neonatal vomiting; phenylalanine to hydroxylase mental retardation tyrosine
13 Amino Acid Degradation: Overview We learned that: • amino acids from protein are an important energy source in carnivorous animals • the first step of AA catabolism is transfer of the NH3 via PLP-dependent aminotransferase usually to a-ketoglutarate to yield L-glutamate • in most mammals, toxic ammonia is quickly recaptured into carbamoyl phosphate and passed into the urea cycle • amino acids are degraded to pyruvate, acetyl-CoA, α- ketoglutarate, succinyl-CoA, and/or oxaloacetate • amino acids yielding acetyl-CoA are ketogenic • amino acids yielding other end products are glucogenic • genetic defects in amino degradation pathways result in a number of human diseases • amino acid catabolism is dependent on a variety of cofactors, including THF, ado-Met (SAM), Cbl, biotin, and PLP
End of Material for Exam 3
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