February 14 Chapter 26 Amino Acid Metabolism Biochemistry 432/832 February 14 Chapter 26 Amino Acid Metabolism

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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

The nitrogen cycle

The glutamate dehydrogenase reaction

The glutamine synthetase reaction

The glutamate dehydrogenase/glutamine synthase pathway Two N fixing steps - one inefficient One each

The glutamate synthase reaction

The glutamine synthase/GOGAT pathway One N fixing step - inefficient but expensive One NADPH Two ATP

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

Allosteric regulation of glutamine synthase activity by feedback inhibition

Covalent Modification of Glutamine Synthetase Each subunit is adenylylated at Tyr-397 Adenylylation inactivates GS Adenylyl transferase catalyzes both the adenylylation and deadenylylation PII (regulatory protein) controls both activities AT:PIIA catalyzes adenylylation AT:PIID catalyzes deadenylylation -ketoglutarate and Gln regulate PII

Covalent modification of glutamine synthase - adenylylation of Tyr397

The cyclic cascade system that regulates the covalent modification of GS

Gene Expression regulates GS Gene GlnA is actively transcribed only if transcriptional enhancer NRI is in its phosphorylated form, NRI-P NRI is phosphorylated by NRII, a protein kinase If NRII is complexed with PIIA (inactivator) it acts as a phosphatase, not a kinase

Transcriptional regulation of GlnA expression through the reversible phosphorylation of NR1 Activated by glutamine, inactivated by a-ketoglutarate

Amino Acid Biosynthesis Plants and microorganisms can make all 20 amino acids and all other organisms need N metabolites In these organisms, glutamate is the source of N, via transamination (aminotransferase) reactions Mammals can make only 10 of the 20 amino acids 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

Glutamate-dependent transamination - primary mechanism for amino acid synthesis

The -Ketoglutarate Family Glu, Gln, Pro, Arg, and sometimes Lys

Proline biosynthesis from glutamate

Synthesis of ornithine from glutamate - a step in arginine biosynthesis

The Urea Cycle N and C in the guanidino group of Arg come from NH4+, HCO3- (carbamoyl-P), and the -NH2 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

The urea cycle

The Aspartate Family Asp, Asn, Lys, Met, Thr, Ile Transamination of oxaloacetate gives Asp Amidation of Asp gives Asn Thr, Met, and Lys are made from Asp

Transamination of oxaloacetate yields aspartate

Asp + Gln --> Asn + Glu

The Pyruvate Family Ala, Val, Leu Transamination of pyruvate gives Ala Val is derived from pyruvate Leu synthesis, like that of Ile and Val, begins with an -keto acid Transaminations from Glu complete each of these pathways

3-Phosphoglycerate Family Ser, Gly, Cys 3-Phosphoglycerate dehydrogenase diverts 3-PG from glycolysis to amino acid paths Transamination by Glu gives 3-P-serine Phosphatase yields serine A PLP-dependent enzyme makes Cys

Biosynthesis of serine from 3-phosphoglycerate

Glycine biosynthesis from serine

Cysteine biosynthesis Sulfhydration of serine by sulfide Sulfhydration of O-acetylserine

Sulfate assimilation

Degradation of Amino Acids The 20 amino acids are degraded to produce TCA and glycolytic intermediates

Degradation of amino acids

Degradation of Ala, Ser, Cys, Gly and Trp and Thr to pyruvate