Analysis of the mechanisms for uronate isomerase from E. coli, cobyrinic acid a,c-diamide synthetase from S. typhimurium, and cobyric acid synthetase from S. typhimurium.

摘要

Uronate isomerase catalyzes the isomerization of D-glucuronate and Dfructuronate.This enzyme has been classified as a member of the amidohydrolasesuperfamily. The reaction catalyzed by uronate isomerase is analogous to theisomerization of aldose/ketose sugars. These interconversions can occur via twomechanisms, a hydride or proton transfer. The solvent exchange experiments and theelimination of fluoride from 3-deoxy-3-fluoro-D-glucuronate catalyzed by the enzymesupport a proton transfer. Assignment of the transferred proton as the proR proton furthersupports a proton transfer mechanism via a cis-enediol intermediate for uronateisomerase from E. coli.Cobyrinic acid a,c-diamide synthetase and cobyric acid synthetase from S.typhimurium catalyze ATP dependent amidations of carboxylate groups on the peripheryof cobyrinic acid utilizing glutamine or ammonia as a nitrogen donor. The role of ATP inthe reaction has been probed by positional isotope exchange (PIX). The results confirm the presence of phosphorylated intermediate species in the reactions catalyzed bycobyrinic acid a,c-diamide synthetase and cobyric acid synthetase from S. typhimurium.Cobyric acid synthetase catalyzes the amidation of carboxylate groups b, d, e, andg of adenosyl-cobyrinic acid a,c-diamide in the biosynthetic pathway for coenzyme B12.Analysis of the reaction time courses demonstrate the appearance of three uniqueintermediate species which are released from the active site after each amidationreaction. The identification of the intermediate species was accomplished by 1H, 15NHSQC NMR spectroscopy. The NMR spectrum of a sample quenched at the beginningof the reaction shows a single intermediate species corresponding to carboxamide e.Subsequent spectra establish the amidation order as e, d, b, and g. The structural basisfor the dissociative and sequential reaction mechanism coupled with the rigidregiochemistry is unknown. However, mutations to aspartate 146 perturb the order ofamidation. A NMR spectrum quenched early in the reaction with the D146N mutantshows two intermediate species corresponding to carboxamides e and d. Spectra ofsamples later in the reaction confirm the presence of multiple e, d, and g amide species.The reaction is completed with the amidation of carboxylate b.