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 glutamate dehydrogenase/glutamine The glutamate dehydrogenase/glutamine synthase pathwaysynthase pathway
One each
Two N fixing steps - one inefficient
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
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
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 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
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
Sulfhydration of serine by sulfideSulfhydration of serine by sulfide
Sulfhydration of O-acetylserineSulfhydration of O-acetylserine
Cysteine biosynthesisCysteine biosynthesis
Degradation of Amino Acids
The 20 amino acids are degraded to produce TCA and glycolytic intermediates