Nip>2 + p>-activated glyoxalase I from Escherichia coli: Substrate specificity, kinetic isotope effects and evolution within the 尾伪尾尾尾 superfamily
- 作者：Kadia Y. Mullings
- 关键词：Glyoxalase ; Escherichia coli ; Thiol cofactor ; Substrate specificity ; Kinetic isotope effect ; Fosfomycin resistance protein
- 刊名：Journal of Inorganic Biochemistry
- 出版年：2012
- 期刊代码：53_01620134
- 类别：ch
- 出版时间：March, 2012
- 卷：108
- 期：Complete
- 页码：133-140
- 文件大小：751 K
摘要
The Escherichia coli glyoxalase system consists of the metalloenzymes glyoxalase I and glyoxalase II. Little is known regarding Nip>2 + p>-activated E. coli glyoxalase I substrate specificity, its thiol cofactor preference, the presence or absence of any substrate kinetic isotope effects on the enzyme mechanism, or whether glyoxalase I might catalyze additional reactions similar to those exhibited by related 尾伪尾尾尾 structural superfamily members. The current investigation has shown that this two-enzyme system is capable of utilizing the thiol cofactors glutathionylspermidine and trypanothione, in addition to the known tripeptide glutathione, to convert substrate methylglyoxal to non-toxic d-lactate in the presence of Nip>2 + p> ion. E. coli glyoxalase I, reconstituted with either Nip>2 + p> or Cdp>2 + p>, was observed to efficiently process deuterated and non-deuterated phenylglyoxal utilizing glutathione as cofactor. Interestingly, a substrate kinetic isotope effect for the Nip>2 + p>-substituted enzyme was not detected; however, the proton transfer step was observed to be partially rate limiting for the Cdp>2 + p>-substituted enzyme. This is the first non-Znp>2 + p>-activated GlxI where a metal ion-dependent kinetic isotope effect using deuterium-labelled substrate has been observed. Attempts to detect a glutathione conjugation reaction with the antibiotic fosfomycin, similar to the reaction catalyzed by the related superfamily member FosA, were unsuccessful when utilizing the E. coli glyoxalase I E56A mutein.