Replacement by site-directed mutagenesis indicates a role for histidine 170 in the glutamine amide transfer function of anthranilate synthase.
Anthranilate synthase is a glutamine amidotransferase that catalyzes the first reaction in tryptophan biosynthesis. Conserved amino acid residues likely to be essential for glutamine-dependent activity were identified by alignment of the glutamine amide transfer domains in four different enzymes: anthranilate synthase component II (AS II), p-aminobenzoate synthase component II, GMP synthetase, and carbamoyl-P synthetase. Conserved amino acids were mainly localized in three clusters. A single conserved histidine, AS II His-170, was replaced by tyrosine using site-directed mutagenesis. Glutamine-dependent enzyme activity was undetectable in the Tyr-170 mutant, whereas the NH3-dependent activity was unchanged. Affinity labeling of AS II active site Cys-84 by 6-diazo-5-oxonorleucine was used to distinguish whether His-170 has a role in formation or in breakdown of the covalent glutaminyl-Cys-84 intermediate. The data favor the interpretation that His-170 functions as a general base to promote glutaminylation of Cys-84. Reversion analysis was consistent with a proposed role of His-170 in catalysis as opposed to a structural function. These experiments demonstrate the application of combining sequence analyses to identify conserved, possibly functional amino acids, site-directed mutagenesis to replace candidate amino acids, and protein chemistry for analysis of mutationally altered proteins, a regimen that can provide new insights into enzyme function.
D000878:Anthranilate Synthase / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D005796:Genes; D005798:Genes, Bacterial; D005838:Genotype; D005972:Glutaminase; D005973:Glutamine / Q000378:metabolism; D006639:Histidine; D007700:Kinetics; D009154:Mutation; D009838:Oligodeoxyribonucleotides / Q000494:pharmacology; D010957:Plasmids; D012706:Serratia marcescens / Q000201:enzymology* / Q000235:genetics
D009838:Oligodeoxyribonucleotides; D005973:Glutamine; D006639:Histidine; D005972:Glutaminase; D000878:Anthranilate Synthase