Studies of the mechanism of anthranilate synthase reaction

The enzyme anthranilate synthase catalyzes the formation of anthranilate form either chorismate and glutamine or chorismate and ammonia. In the aromatization of chorismate, a hydroxyl group and an enolpyruvyl group must be eliminated. Elimination of the enolpyruvyl group of chorismate is accompanied by protonation to form pyruvate. The source of this proton was investigated by performing the enzymatic reaction in 99.7 per cent D(2)O. The isolated pyruvate contained close to an atom of deuterium in the methyl group. High resolution mass spectra also revealed that about 6 per cent of the deuterio pyruvate contains a -CHD(2) species. Thus, the results obtained conclusively demonstrate that in the formation of the pyruvate, the third hydrogen of the methyl group arises from water and not by intramolecular shift of a hydrogen from the ring of chorismate.