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Transcript
Welcome to Class 14! Introductory Biochemistry! Class 14: Outline and Objectives Overview of amino acid catabolism! • 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! 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! Signals TCA is slowed and need some anaplerotic reactions! The urea cycle looks expensive! Ammonia buildup signal! Overall:! NH4+ + HCO3– + Aspartate + 3 ATP + H2O! !↓! Urea + Fumarate + 2 ADP + AMP + 4 Pi + 5 H+! 4 ATP equivalents! OAA → Aspartate! Malate + NAD+ → OAA + NADH! But:! Fumarate → Malate! Ox. Phosphorylation! ~2.5 ATP! The net cost is ~1.5 ATP! Figure 18-13! Fates of deaminated carbon skeletons! Roles of cofactors in amino acid catabolism! Cofactor !Type of Reaction! ! PLP ! !transamination, ! ! !decarboxylation, ! ! !deamination! Biotin ! !one-carbon transfer (CO2)! THF !one-carbon transfer ! ! ! !(intermediate oxidation states)! AdoMet! !one-carbon transfer (CH3-)! 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! Amino acid biosynthesis! Catabolic pathway for Asn and Asp! Ammonia is incorporated into biomolecules through glutamate and glutamine! ! ! ! Figure 18-8! ! ! ! ! Glutamate + NH4+ + ATP → Glutamine + ADP + Pi + H+! O! This amidation is carried out by Glutamine Synthetase! Figure 18-29! Glutamine synthetase is also regulated through covalent modification! Glutamine synthetase:! a primary regulatory point ! for nitrogen metabolism! Adenylylation of Tyr397! Allosteric Regulators:! His! Feedback! Trp! Inhibitors! Carbamoyl-P! CTP! AMP! Reflect! Metabolic! Gly! Status! Ala! These inhibitors work additively (they are all potential sources of N)! Glutamate synthase (Plants only):! α-Ketoglutarate + glutamine + NADPH + H+ Inactive Gln Synthetase! Active Gln Synthetase! (less sensitive to inhibitors)! Adenylyltransferase! (more sensitive to inhibitors)! Uridylyltransferase! Net Result: ! Gln Synthetase is! 2 glutamate + NADP+! Figure 22-8! More active at high [αKG], high [ATP], low [Gln], low [Pi] ! Less active at low [αKG], low [ATP], high [Gln], high [Pi] ! αKG! ATP! Gln! Pi! 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! Example: 3-phosphoglycerate as a precursor of Ser and Gly! amino groups from Glu by transamination! ! p. 892! Example: 3-phosphoglycerate as a precursor of Ser and Gly! 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:! Specific mechanisms:! • allosteric regulation! • feedback inhibition! concerted! sequential! ● 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! • enzyme multiplicity! Figure 22-11! Figure 22-24! 13! The nitrogen cycle! Denitrification:! Through the reactions of nitrification and denitrification, eventually, all nitrogen in the biosphere would be converted to N2, if it were not for:! anaerobic reduction ! of NO3– to N2! Nitrogen Fixation:! atmospheric dinitrogen (N2) is reduced to a biologically! useful form (NH4+)! + – 0 N2 + 6 H + 6 e → 2 NH3! ΔG' = -33.5 kJ/mol! N2 bond strength is 930 kJ/mol! Nitrification:! Oxidation of NH3 to NO3-! Many free-living aerobic bacteria obtain their energy by oxidizing reduced forms of nitrogen.! ! • Nitrogen fixation is an exergonic reaction! • Under standard conditions, the forward direction is strongly favored! • 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. N2 bond strength is 930 kJ/mol.! • Biological nitrogen fixation occurs at atmospheric pressure and room temperature. How is this accomplished?! Overall reaction:! NH3 + 1.5 O2 → NO2– + H+ + H2O! ΔG’ o = –380.1 kJ/mol! Figure 22-1! Dinitrogenase reductase/dinitrogenase complex! Dinitrogenase reductase! N2 + 6 H+ + 6 e– → 2 NH3! Fdred + Dinitrogenaseox + 2 ATP → Fdox + Dinitrogenasered + 2 ADP + 2 Pi! Dinitrogenase α subunits! • Two ATP are hydrolyzed for each electron conveyed from ferredoxin to dinitrogenase! Dinitrogenase β subunits! • The enzyme is a homodimer! • Each native homodimer contains a single covalently-bound 4Fe/4S cofactor! Dinitrogenase reductase! ➊ Dinitrogen! ➋ A source of electrons (Ferredoxin)! Figure 22-3a! ➌ Lots of energy (ATP hydrolysis)! ➍ Absence or very low concentration of O2 (it inactivates the enzymes)! ➎ Two enzymes that work in series:! 4Fe:4S! ADP! ➀ Dinitrogenase Reductase conveys electrons one at a time from the initial electron source to dinitrogenase, in an ATP-requiring reaction! ➁ Dintrogenase uses the electrons obtained from Dinitrogenase Reductase to reduce nitrogen to ammonia! 14! The nitrogenase complex has a transient existence! Dinitrogenase! Dinitrogenasered + N2 → Dinitrogenaseox + 2 NH3! Electrons! from pyruvate! • The enzyme is an α2β2 heterotetramer! • Each native heterotetramer contains two each of two covalently-bound cofactors, called P clusters and M clusters! M cluster! P cluster! One electron at a time is transferred from ferredoxin to dinitrogenase reductase! Reduction of! dinitrogenase! reductase! The complex between dinitrogenase reductase and dinitrogenase forms and dissociates each time one electron is transferred between them! Reduction of! dinitrogenase! M cluster! Reduction of N2 ! Figure 22-3b! 2e-! Figure 22-4! 15!
