Characterization of the glutamine site of Escherichia coli guanosine 5'-monophosphate synthetase.
Alkylation of guanosine 5'-monophosphate (GMP) synthetase with the glutamine analogs L-2-amino-4-oxo-5-chloropentanoic acid (chloroketon) and 6-diazo-5-oxonorleucine (DON) inactivated glutamine- and NH3-dependent GMP synthetase. Inactivation exhibited second order kinetics. Complete inactivation was accompanied by covalent attachment of 0.4 to 0.5 equivalent of chloroketon/subunit. Alkylation of GMP synthetase with iodacetamide selectively inactivated glutamine-dependent activity. The NH3-dependent activity was relatively unaffected. Approximately 1 equivalent of carboxamidomethyl group was incorporated per subunit. Carboxymethylcysteine was the only modified amino acid hydrolysis. Prior treatment with chloroketone decreased the capacity for alkylation by iodacetamide, suggesting that both reagents alkylate the  same residue. GMP synthetase exhibits glutaminase activity when ATP is replaced by adenosine plus PPi. Iodoacetamide inactivates glutaminase concomitant with glutamine-dependent GMP synthetase. Analysis of pH versus velocity and Km data indicates that the amide of glutamine remains enzyme bound and does not mix with exogenous NH3 in the synthesis of GMP.
D000478:Alkylation; D000596:Amino Acids / Q000494:pharmacology; D000641:Ammonia / Q000494:pharmacology; D019731:Carbon-Nitrogen Ligases; D003980:Diazooxonorleucine / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology*; D005972:Glutaminase / Q000037:antagonists & inhibitors; D005973:Glutamine / Q000031:analogs & derivatives / Q000494:pharmacology*; D006150:Guanine Nucleotides; D006863:Hydrogen-Ion Concentration; D007460:Iodoacetamide / Q000494:pharmacology; D007700:Kinetics; D008025:Ligases / Q000037:antagonists & inhibitors / Q000378:metabolism; D010421:Pentanoic Acids / Q000494:pharmacology; D011485:Protein Binding
D000596:Amino Acids; D006150:Guanine Nucleotides; D010421:Pentanoic Acids; D003980:Diazooxonorleucine; D005973:Glutamine; D000641:Ammonia; D005972:Glutaminase; D008025:Ligases; D019731:Carbon-Nitrogen Ligases; C020828:GMP synthase (glutamine-hydrolyzing); D007460:Iodoacetamide