Biochemistry 432/832

February 14February 14

Chapter 26Chapter 26

Amino Acid Metabolism

Announcements:

-

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 cycleThe nitrogen cycle

The glutamate dehydrogenase reactionThe glutamate dehydrogenase reaction

The glutamine synthetase reactionThe glutamine synthetase reaction

The glutamate dehydrogenase/glutamine The glutamate dehydrogenase/glutamine synthase pathwaysynthase pathway

One each

Two N fixing steps - one inefficient

The glutamate synthase reactionThe glutamate synthase reaction

The glutamine synthase/GOGAT pathwayThe glutamine synthase/GOGAT pathway

One NADPH

Two ATP

One N fixing step - inefficient but expensive

Glutamine SynthetaseA 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 Modificationof 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 - Covalent modification of glutamine synthase - adenylylation of Tyr397adenylylation of Tyr397

The cyclic cascade system that regulates the The cyclic cascade system that regulates the covalent modification of GScovalent modification of GS

Gene Expressionregulates 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 Transcriptional regulation of GlnA expression through the reversible phosphorylation of NR1through the reversible phosphorylation of NR1

Activated by glutamine, inactivated by -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 Glutamate-dependent transamination - primary mechanism for amino acid synthesismechanism for amino acid synthesis

The -Ketoglutarate Family

Glu, Gln, Pro, Arg, and sometimes Lys

Proline biosynthesis from glutamateProline biosynthesis from glutamate

Synthesis of ornithine from glutamate - a step Synthesis of ornithine from glutamate - a step in arginine biosynthesisin 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 The urea cyclecycle

The Aspartate FamilyAsp, 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 Transamination of oxaloacetate yields aspartateaspartate

Asp + Gln --> Asn + GluAsp + Gln --> Asn + Glu

The Pyruvate FamilyAla, 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 FamilySer, 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 Biosynthesis of serine from 3-serine from 3-phosphoglyceratephosphoglycerate

Glycine biosynthesis from serineGlycine biosynthesis from serine

Sulfhydration of serine by sulfideSulfhydration of serine by sulfide

Sulfhydration of O-acetylserineSulfhydration of O-acetylserine

Cysteine biosynthesisCysteine biosynthesis

Sulfate Sulfate assimilationassimilation

Degradation of Amino Acids

The 20 amino acids are degraded to produce TCA and glycolytic intermediates

Degradation of Degradation of amino acidsamino acids

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