Glutamine amidotransferase function. Replacement of the active-site cysteine in glutamine phosphoribosylpyrophosphate amidotransferase by site-directed mutagenesis.
Site-directed mutagenesis was employed to replace cysteine 12 with phenylalanine in Bacillus subtilis glutamine phosphoribosylpyrophosphate amidotransferase (amidophosphoribosyltransferase). Glutamine-dependent amidophosphoribosyltransferase activity was abolished as a consequence of the mutation. The mutant enzyme, however, exhibited NH3-dependent activity, contained Fe-S, and was normally regulated by AMP. These results document the role of the active site cysteine in activation of glutamine for amide transfer. NH3-dependent amidophosphoribosyltransferase was utilized for de novo purine nucleotide synthesis. Cells containing the mutant enzyme grew at nearly the wild-type rate in media containing a high concentration of NH4Cl. The Phe-12 mutation was used to study NH2-terminal processing. Whereas the wild-type Cys-12 enzyme is processed correctly in Escherichia coli by removal of 11 amino acid residues from the NH2 terminus, the Phe-12 mutant enzyme was not subject to undecapeptide processing. Neither the mutant nor wild-type enzyme made in vitro was correctly processed. Alternative enzymatic and autocatalytic processing mechanisms were considered. The available evidence favors autocatalytic NH2-terminal undecapeptide processing.
D000249:Adenosine Monophosphate / Q000494:pharmacology; D000595:Amino Acid Sequence; D000878:Anthranilate Synthase; D001412:Bacillus subtilis / Q000201:enzymology / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D003545:Cysteine / Q000378:metabolism*; D004262:DNA Restriction Enzymes / Q000378:metabolism; D009154:Mutation; D019884:Nitrogenous Group Transferases; D010649:Phenylalanine / Q000378:metabolism; D014166:Transferases / Q000235:genetics / Q000378:metabolism*
D000249:Adenosine Monophosphate; D010649:Phenylalanine; D014166:Transferases; D019884:Nitrogenous Group Transferases; D004262:DNA Restriction Enzymes; D000878:Anthranilate Synthase; C020655:anthranilate synthase, glutamine amidotransferase subunit; D003545:Cysteine