Kinetic Characterization of Wild-Type and S229A Mutant MurB: Evidence for the Role of Ser 229 as a General Acid

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• 作者：Timothy E. Benson ; Christopher T. Walsh ; and Vincent Massey
• 刊名：Biochemistry
• 出版年：1997
• 出版时间：January 28, 1997
• 年：1997
• 卷：36
• 期：4
• 页码：796 - 805
• 全文大小：527K
• 年卷期：v.36,no.4(January 28, 1997)
• ISSN：1520-4995

文摘

X-ray derived structural data predicted that serine 229 waspositioned to act as a proton donorto the developing C2 carbanion during the reduction ofenolpyruvyl-UDP-N-acetylglucosamine catalyzedby the bacterial peptidoglycan biosynthetic flavoenzyme MurB. Toinvestigate this effect, a mutant whereserine 229 was replaced by alanine was constructed and purified.Kinetic analysis of the two half-reactionsfor the mutant enzyme revealed a 9-fold decrease in the reduction ofEFl_ox by NADPH and a dramatic10⁷-fold decrease in the reoxidation of EFl_redwith the enolpyruvyl substrate. In addition, studies ofS229Awith the substrate analog,(E)-enolbutyryl-UDP-N-acetylglucosamine, showed astriking bias of thepartitioning toward formation of the (Z) geometric isomer asopposed to formation of the reduced productUDP-methylmuramic acid, which was the predominant product in wild-typeMurB. These studies provideevidence for the proposed role of this active-site serine as a generalacid catalyst.