Anthranilate synthase from Bacillus subtilis. The role of a reduced subunit X in aggregate formation and amidotransferase activity.
With respect to its sulfhydryl groups, subunit X can exist in at least two forms, oxidized (Xox) and reduced (Xre). The importance of the Xre form for the formation of an EX complex and for amidotransferase activity has been examined. Subunit Xre is rapidly inactivated by p-chloromercuribenzoate and bromopyruvate, whereas subunit Xox, which is not catalytically functional in amidotransferase activity, is not affected. The glutamine analogue 6-diazo-5-oxo-L-norleucine (DON) has no effect on Xre alone but rapidly inactivates the EXre complex. DON-inactivated subunit X cannot be reactivated by 2-mercaptoethanol but can be readily displaced from subunit E by free subunit Xre. The integrity of the EXre complex is maintained following gel filtration on Sephadex G-100 in the presence of glutamine and 2-mercaptoethanol, thus the binding of glutamine to the complex does not require the binding of other substrates. Subunit Xox, however, does not aggregate with subunit E since no EXox complex is found following gel filtration on Sephadex G-100 in the presence of glutamine and in the absence of 2-mercaptoethanol. Thus, a reduced sulfhydryl group(s) is not only essential for amidotransferase activity but also for the formation of the aggregate as well. The following model is proposed to explain these results. Free subunit Xre does not bind DON or glutamine to the catalytically functional sulfhydryl group. Upon aggregation with subunit E, however, the glutamine or DON binds to the glutamine catalytic site on subunit Xre and amidotransfer or alkylation occurs. An EX complex which has been alkylated by DON can be readily dissociated and it is suggested that following catalysis the EX complex may also dissociate.
D000577:Amides / Q000378:metabolism*; D000619:Aminohydrolases / Q000378:metabolism; D000878:Anthranilate Synthase / Q000302:isolation & purification / Q000378:metabolism; D001391:Azo Compounds / Q000494:pharmacology; D001412:Bacillus subtilis / Q000201:enzymology*; D002729:Chloromercuribenzoates / Q000494:pharmacology; D002827:Chorismic Acid / Q000494:pharmacology; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004355:Drug Stability; D004591:Electrophoresis, Polyacrylamide Gel; D005973:Glutamine / Q000031:analogs & derivatives / Q000494:pharmacology; D007700:Kinetics; D046911:Macromolecular Substances; D008274:Magnesium / Q000494:pharmacology; D008623:Mercaptoethanol / Q000494:pharmacology; D009154:Mutation; D009646:Norleucine / Q000494:pharmacology; D013997:Time Factors
D000577:Amides; D001391:Azo Compounds; D002729:Chloromercuribenzoates; D046911:Macromolecular Substances; D005973:Glutamine; D008623:Mercaptoethanol; D009646:Norleucine; D000619:Aminohydrolases; D000878:Anthranilate Synthase; D002827:Chorismic Acid; D008274:Magnesium