Welcome to Class 14 Introductory Biochemistry Class 14: Outline and Objectives Amino Acid Catabolism Fates of amino groups transamination urea cycle Fates of carbon skeletons important cofactors metabolic products glucogenic & ketogenic amino acids Errors of metabolism Incorporation of Ammonia into Biomolecules Glutamine synthetase Amino Acid Biosynthesis Biosynthetic families Regulation Nitrogen Interconversions Overview of amino acid catabolism Figure 18-1 1
Transamination Fates of amino groups Transamination Carried out by aminotransferases amino group transfer: amino acid α-keto acid Ammonia is toxic There is no net gain or loss of NH3 or electrons Figure 18-2a Structure: pyridoxal phosphate prosthetic group Figure 18-4 Mechanism: pyridoxal phosphate prosthetic group PLP PMP: acts as an amino group carrier Figure 18-5a, b Figure 18-6a 2
In active muscle, alanine is used to transport ammonia: NH4+ Transport to Liver: Glu is charged and cannot easily pass through cell membranes, and ammonia is toxic As elsewhere, α-kg + AA α-ka + Glu This is OK in the liver What about in other tissues? Elsewhere, Glu + NH4+ Gln and, Glu + NH4+ Gln is possible other tissues But, muscle glycolysis pyruvate (lots) So, by transamination: Pyr + Glu α-kg + Ala Liver Gln transports ammonia in the bloodstream to the liver, where... Gln Glu + NH4+ Figure 18-9 Figure 18-8 In liver: oxidative deamination of glutamate to generate ammonia Fates of amino groups Net loss of NH3 from amino acids requires oxidation: α-keto acids are 2 equivalents more oxidized than amino acids End result: Amino groups are collected in the liver Glutamate Dehydrogenase Figure 18-7 Most amino acids pass their amino groups through glutamate Figure 18-2a 3
NH3 enters the urea cycle via carbamoyl phosphate, which is formed from NH4+ and HCO3 Major excretory forms of nitrogen alkaline, accumulation raises ph, used mostly by aquatic animals neutral, but excretion requires high H2O loss This step is rate limiting activated carbamoyl group donor ATP activates bicarbonate acquisition of first N insoluble, excretion can occur with low H2O loss 2nd ATP phosphorylates carbamate This reaction is catalyzed by Carbamoyl Phosphate Synthetase I (CPS I), a key regulatory enzyme. CPS I is a mitochondrial matrix enzyme How is urea generated from NH4+? Figure 18-2b The urea cycle occurs partly in the cytoplasm and partly in the mitochondria Figure 18-11 Linkage between the urea cycle and the citric acid cycle from Glycolysis ① carbamoyl-p + L-ornithine L-citrulline ② L-citrulline + L-aspartate argininosuccinate (acquisition of 2nd N) OAA OAA ③ argininosuccinate L-arginine + fumarate ④ hydrolysis of L-arginine to form urea NH3 Figure 18-10 Figure 18-12 4
Regulation of the flux through the urea cycle The urea cycle looks expensive Signals TCA is slowed and need some anaplerotic reactions Ammonia buildup signal Overall: NH 4 + + HCO 3 + Aspartate + 3 ATP + H 2 O Urea + Fumarate + 2 ADP + AMP + 4 P i + 5 H + 4 ATP equivalents Figure 18-13 OAA Aspartate But: Malate + NAD + OAA + NADH Ox. Phosphorylation Fumarate Malate ~2.5 ATP The net cost is ~1.5 ATP Fates of deaminated carbon skeletons Roles of cofactors in amino acid catabolism Cofactor Type of Reaction PLP transamination, decarboxylation, deamination Biotin one-carbon transfer (CO 2 ) THF one-carbon transfer (intermediate oxidation states) AdoMet one-carbon transfer (CH 3 -) THB oxidation/reduction Figure 18-15 5
Biotin transfers CO2 AdoMet transfers CH3 CO2 binds here protein binds here Figure 18-16 CO2 + R + biotin-protein CO2-biotin-protein + R biotin-protein + R-CO2 THF transfers carbons in intermediate oxidation states Figure 18-16 Figure 18-18 Fates of deaminated carbon skeletons Vitamin B-9 Figure 18-16 Figure 18-17 Figure 18-15 6
Catabolic pathway for Thr and Gly Catabolic pathway for Trp, Ala, Ser, and Cys 2 Figure 18-19 Fates of deaminated carbon skeletons Figure 18-19 Catabolic pathway for Tyr, and Phe Figure 18-15 Figure 18-23 7
Catabolic pathway for Tyr, and Phe Fates of deaminated carbon skeletons Figure 18-15 Figure 18-23 Catabolic pathway for Val, Ile, Leu Fates of deaminated carbon skeletons TPP, FAD, lipoate Sotolone Figure 18-28 Figure 18-15 8
Catabolic pathway for Asn and Asp Amino acid biosynthesis Ammonia is incorporated into biomolecules through glutamate and glutamine Figure 18-8 O Glutamate + NH 4 + + ATP Glutamine + ADP + P i + H + Figure 18-29 This amidation is carried out by Glutamine Synthetase Glutamine synthetase: a primary regulatory point for nitrogen metabolism Allosteric Regulators: His Trp Feedback Carbamoyl-P Inhibitors CTP AMP Gly Ala Reflect Metabolic Status These inhibitors work additively (they are all potential sources of N) Glutamate synthase (Plants only): α-ketoglutarate + glutamine + NADPH + H + 2 glutamate + NADP + Figure 22-8 Glutamine synthetase is also regulated through covalent modification Adenylylation of Tyr 397 Active Gln Synthetase (less sensitive to inhibitors) Net Result: Gln Synthetase is Adenylyltransferase More active at high [αkg], high [ATP], low [Gln], low [P i ] Less active at low [αkg], low [ATP], high [Gln], high [P i ] Inactive Gln Synthetase (more sensitive to inhibitors) Uridylyltransferase αkg ATP Gln P i Figure 22-9a 9
Amino acids can be grouped according to their carbon skeleton precursors Glutamine amidotransferases Transfer of glutamine amido nitrogen to hydroxyl compounds: So, through glutamine synthetase: Glutamate + NH4+ + ATP glutamine + ADP + Pi + H+ Mammals can synthesize only about half of all amino acids. Then, through glutamine amidotransferases: glutamine + R-OH glutamate + R-NH2 Figure 22-10 Overview of amino acid biosynthesis Overview of amino acid biosynthesis Precursors from: Glycolysis Citric Acid Cycle Pentose Phosphate Pathway Figure 22-11 Figure 22-11 10
Some amino acids are derived from α-ketoglutarate Some amino acids are derived from 3-phosphoglycerate p. 892 p. 892 Example: 3-phosphoglycerate as a precursor of Ser and Gly Example: 3-phosphoglycerate as a precursor of Ser and Gly amino groups from Glu by transamination one-carbon transfer using THF Figure 22-14 Figure 22-14 11
Biosynthesis of Cys in mammals Biosynthesis of Cys in mammals Although vertebrates cannot synthesize Met, they can use the sulfur atom of Met in the synthesis of Cys Figure 18-18 Figure 22-16 Several amino acids are derived from oxaloacetate and from pyruvate Aromatic amino acids are synthesized from PEP & erythrose 4-phosphate through a common precursor Citric Acid Cycle Themes: energy input ATP use of reducing power NAD(P)H amino groups from Glu by transamination one-carbon transfer using THF p. 898 Glycolysis OAA Asp Pyr Ala direct transamination direct transamination p. 895 Phenylalanine and tryptophan are essential amino acids for vertebrates we can t do this 12
Histidine is derived from: Summary of amino acid biosynthesis: 6 biosynthetic families PRPP ATP Gln Glu Figure 22-22 The 5-phosphoribosyl-1-pyrophosphate precursor is synthesized from ribose 5phosphate PRPP is an intermediate in the biosynthesis of: Trp His Nucleotides Histidine is an essential amino acid for vertebrates we can t do this Precursors from: Glycolysis Citric Acid Cycle Pentose Phosphate Pathway p. 892 p. 898a, b Summary of amino acid biosynthesis Regulatory control of amino acid biosynthesis Regulation can be complex: levels of all AAs must be balanced, so synthesis must be coordinated many AAs share common precursors, so simple feedback inhibition could shut down the synthesis of several AAs Specific mechanisms: allosteric regulation feedback inhibition concerted sequential enzyme multiplicity Figure 22-11 Figure 22-24 13
The nitrogen cycle Denitrification: anaerobic reduction of NO 3 to N 2 Through the reactions of nitrification and denitrification, eventually, all nitrogen in the biosphere would be converted to N 2, if it were not for: Nitrogen Fixation: atmospheric dinitrogen (N 2 ) is reduced to a biologically useful form (NH 4+ ) N 2 + 6 H + + 6 e 2 NH 3 Nitrogen fixation is an exergonic reaction N 2 bond strength is 930 kj/mol Under standard conditions, the forward direction is strongly favored ΔG' 0 = -33.5 kj/mol Nitrification: Oxidation of NH 3 to NO 3 - Many free-living aerobic bacteria obtain their energy by oxidizing reduced forms of nitrogen. Overall reaction: NH 3 + 1.5 O 2 NO 2 + H + + H 2 O ΔG o = 380.1 kj/mol Figure 22-1 However, non-biological nitrogen fixation by the industrial Haber-Bosch process requires about 400 C and several hundred atmospheres of pressure. Why? Although the reaction is thermodynamically favorable, it is kinetically very unfavorable, i.e., it has a very large positive ΔG. The uncatalyzed reaction is infinitesimally slow. N 2 bond strength is 930 kj/mol. Biological nitrogen fixation occurs at atmospheric pressure and room temperature. How is this accomplished? ➊ ➋ ➌ ➍ ➎ Dinitrogenase reductase/dinitrogenase complex Dinitrogen A source of electrons (Ferredoxin) Lots of energy (ATP hydrolysis) Absence or very low concentration of O 2 (it inactivates the enzymes) Two enzymes that work in series: ➀ ➁ N 2 + 6 H + + 6 e 2 NH 3 Dinitrogenase α subunits Dinitrogenase β subunits Dinitrogenase reductase Figure 22-3a Dinitrogenase Reductase conveys electrons one at a time from the initial electron source to dinitrogenase, in an ATP-requiring reaction Dintrogenase uses the electrons obtained from Dinitrogenase Reductase to reduce nitrogen to ammonia Dinitrogenase reductase Fd red + Dinitrogenase ox + 2 ATP Fd ox + Dinitrogenase red + 2 ADP + 2 P i Two ATP are hydrolyzed for each electron conveyed from ferredoxin to dinitrogenase The enzyme is a homodimer Each native homodimer contains a single covalently-bound 4Fe/4S cofactor 4Fe:4S ADP 14
Dinitrogenase The nitrogenase complex has a transient existence Dinitrogenase red + N 2 Dinitrogenase ox + 2 NH 3 The enzyme is an α 2 β 2 heterotetramer Each native heterotetramer contains two each of two covalently-bound cofactors, called P clusters and M clusters P cluster M cluster Electrons from pyruvate Reduction of dinitrogenase reductase One electron at a time is transferred from ferredoxin to dinitrogenase reductase M cluster Reduction of dinitrogenase The complex between dinitrogenase reductase and dinitrogenase forms and dissociates each time one electron is transferred between them Reduction of N 2 2e - Figure 22-3b Figure 22-4 15