Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Chapter 26 Nitrogen Acquisition and Amino Acid Metabolism to accompany Biochemistry, 2/e by Reginald Garrett and Charles Grisham All rights reserved. Requests for permission to make copies of any part of the work should be mailed to: Permissions Department, Harcourt Brace & Company, 6277 Sea Harbor Drive, Orlando, Florida
Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Outline 26.1 The Two Major Pathways of N Acquisition 26.2 The Fate of Ammonium 26.3 Glutamine Synthetase 26.4 Amino Acid Biosynthesis 26.5 Metabolic Degradation of Amino Acids
Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Major Pathways for N Acquisition All biological compounds contain N in a reduced form The principal inorganic forms of N are in an oxidized state Thus, N acquisition must involve reduction of the oxidized forms (N 2 and NO 3 - ) to NH 4 + Nearly all of this is in microorganisms and green plants. Animals gain N through diet.
Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Overview of N Acquisition Nitrogen assimilation and nitrogen fixation Nitrate assimilation occurs in two steps: 2e - reduction of nitrate to nitrite and 6e - reduction of nitrite to ammonium (page 854) Nitrate assimilation accounts for 99% of N acquisition by the biosphere Nitrogen fixation involves reduction of N 2 in prokaryotes by nitrogenase
Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Nitrate Assimilation Electrons are transferred from NADH to nitrate Pathway involves -SH of enzyme, FAD, cytochrome b and MoCo - all protein-bound Nitrate reductases are big kD See Figure 26.2 for MoCo structure MoCo required both for reductase activity and for assembly of enzyme subunits to active dimer
Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Nitrite Reductase Light drives reduction of ferredoxins and electrons flow to 4Fe-4S and siroheme and then to nitrite See Figure 26.2b for siroheme structure Nitrite is reduced to ammonium while still bound to siroheme In higher plants, nitrite reductase is in chloroplasts, but nitrate reductase is cytosolic
Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Enzymology of N fixation Only occurs in certain prokaryotes Rhizobia fix nitrogen in symbiotic association with leguminous plants Rhizobia fix N for the plant and plant provides Rhizobia with carbon substrates All nitrogen fixing systems appear to be identical They require nitrogenase, a reductant (reduced ferredoxin), ATP, O-free conditions and regulatory controls (ADP inhibits and NH 4 + inhibits expression of nif genes
Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Nitrogenase Complex Two protein components: nitrogenase reductase and nitrogenase Nitrogenase reductase is a 60 kD homodimer with a single 4Fe-4S cluster Very oxygen-sensitive Binds MgATP 4ATP required per pair of electrons transferred Reduction of N 2 to 2NH 3 + H 2 requires 4 pairs of electrons, so 16 ATP are consumed per N 2
Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Why should nitrogenase need ATP??? N 2 reduction to ammonia is thermodynamically favorable However, the activation barrier for breaking the N-N triple bond is enormous 16 ATP provide the needed activation energy
Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Nitrogenase A 220 kD heterotetramer Each molecule of enzyme contains 2 Mo, 32 Fe, 30 equivalents of acid-labile sulfide (FeS clusters, etc) Four 4Fe-4S clusters plus two FeMoCo, an iron-molybdenum cofactor Nitrogenase is slow - 12 e - pairs per second, i.e., only three molecules of N 2 per second
Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company The Fate of Ammonium Three major reactions in all cells Carbamoyl-phosphate synthetase I –two ATP required - one to activate bicarb, one to phosphorylate carbamate Glutamate dehydrogenase –reductive amination of alpha-ketoglutarate to form glutamate Glutamine synthetase –ATP-dependent amidation of gamma- carboxyl of glutamate to glutamine
Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Ammonium Assimilation Two principal pathways Principal route: GDH/GS in organisms rich in N See Figure both steps assimilate N Secondary route: GS/GOGAT in organisms confronting N limitation GOGAT is glutamate synthase or glutamate:oxo-glutarate amino transferase See Figures and 26.13
Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Glutamine Synthetase A Case Study in Regulation GS in E. coli is regulated in three ways: –Feedback inhibition –Covalent modification (interconverts between inactive and active forms) –Regulation of gene expression and protein synthesis control the amount of GS in cells –But no such regulation occurs in eukaryotic versions of GS
Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Allosteric Regulation of Glutamine Synthetase Nine different feedback inhibitors: Gly, Ala, Ser, His, Trp, CTP, AMP, carbamoyl-P and glucosamine-6-P Gly, Ala, Ser are indicator of amino acid metabolism in cells Other six are end products of a biochemical pathway This effectively controls glutamine’s contributions to metabolism
Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Covalent Modification of Glutamine Synthetase Each subunit is adenylylated at Tyr-397 Adenylylation inactivates GS Adenylyl transferase catalyzes both the adenylylation and deadenylylation P II (regulatory protein) controls these AT:P IIA catalyzes adenylylation AT:P IID catalyzes deadenylylation -ketoglutarate and Gln also affect
Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Gene Expression regulates GS Gene GlnA is actively transcribed only if transcriptional enhancer NR I is in its phosphorylated form, NR I -P NR I is phosphorylated by NR II, a protein kinase If NR II is complexed with P IIA it acts as a phosphatase, not a kinase
Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Amino Acid Biosynthesis Plants and microorganisms can make all 20 amino acids and all other needed N metabolites In these organisms, glutamate is the source of N, via transamination (aminotransferase) reactions Mammals can make only 10 of the 20 aas The others are classed as "essential" amino acids and must be obtained in the diet All amino acids are grouped into families according to the intermediates that they are made from - see Table 26.1
Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company The -Ketoglutarate Family Glu, Gln, Pro, Arg, and sometimes Lys Proline pathway is chemistry you have seen before in various ways Look at ornithine pathway to see the similarity to the proline pathway Note that CPS-I converts ornithine to citrulline in the urea cycle (Figure 26.23) Know the CPS-I mechanism - Figure 26.22
Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company The Urea Cycle N and C in the guanidino group of Arg come from NH 4 +, HCO 3 - (carbamoyl-P), and the -NH 2 of Glu and Asp Breakdown of Arg in the urea cycle releases two N and one C as urea Important N excretion mechanism in livers of terrestrial vertebrates Urea cycle is linked to TCA by fumarate
Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Lysine Biosynthesis in some fungi and in Euglena Lys derived from -ketoglutarate Must add one C - it’s done as in TCA! Transamination gives -aminoadipate Adenylylation activates the -COOH for reduction Reductive amination give saccharopine Oxidative cleavage yields lysine
Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company The Aspartate Family Asp, Asn, Lys, Met, Thr, Ile Transamination of OAA gives Asp Amidation of Asp gives Asn Thr, Met, and Lys are made from Asp (See Figure 26.27) -Aspartyl semialdehyde and homoserine are branch points Note role of methionine in methylations via S-adenosylmethionine (Fig )
Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company The Pyruvate Family Ala, Val, Leu Transamination of pyruvate gives Ala Val is derived from pyruvate Note that Ile synthesis from Thr mimics Val synthesis from pyruvate (Fig ) Leu synthesis, like that of Ile and Val, begins with an -keto acid Transaminations from Glu complete each of these pathways (Figs )
Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 3-Phosphoglycerate Family Ser, Gly, Cys 3-Phosphoglycerate dehydrogenase diverts 3-PG from glycolysis to aa paths Transamination by Glu gives 3-P-serine Phosphatase yields serine Serine hydroxymethylase (PLP) transfers the -carbon of Ser to THF to make glycine A PLP-dependent enzyme makes Cys
Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Aromatic Amino Acids Phe, Tyr, Trp, His Shikimate pathway yields Phe, Tyr, Trp Note the role of chorismate as a branch point in this pathway (Figs ) Note the ‘channeling’ in tryptophan synthase (Figure 26.39) His synthesis, like that of Trp, shares metabolic intermediates with purine biosynthetic pathway
Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Degradation of Amino Acids The 20 amino acids are degraded to produce (mostly) TCA intermediates Know the classifications of amino acids in Figure Know which are glucogenic and ketogenic Know which are purely ketogenic
Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company