Kinetic Analysis of Escherichia coli 2-C-Methyl-D-erythritol-4-phosphate Cytidyltransferase, Wild Type and Mutants, Reveals Roles of Active Site Amino Acids

详细信息    查看全文

• 作者：Sté ; phane B. Richard ; Antonietta M. Lillo ; Charles N. Tetzlaff ; Marianne E. Bowman ; Joseph P. Noel ; and David E. Cane
• 刊名：Biochemistry
• 出版年：2004
• 出版时间：September 28, 2004
• 年：2004
• 卷：43
• 期：38
• 页码：12189 - 12197
• 全文大小：301K
• 年卷期：v.43,no.38(September 28, 2004)
• ISSN：1520-4995

文摘

Escherichia coli 2-C-methyl-D-erythritol-4-phosphate cytidyltransferase (YgbP or IspD)catalyzes the conversion of 2-C-methyl-D-erythritol 4-phosphate (MEP) and cytidine triphosphate (CTP)to 4-diphosphocytidyl-2-C-methylerythritol (CDPME). Pulse chase experiments established that the reactioninvolves an ordered sequential mechanism with mandatory initial binding of CTP. On the basis of analysisof the previously reported crystal structures of apo-YgbP as well as YgbP complexed with both CTP·Mg²⁺ and CDPME·Mg²⁺ [Richard, S. B., Bowman, M. E., Kwiatkowski, W., Kang, I., Chow, C., Lillo,A. M., Cane, D. E., and Noel, J. P. (2001) Nat. Struct. Biol. 8, 641-648], a group of active site residueswere selected for site-directed mutagenesis and steady-state kinetic analysis. Both Lys27 and Lys213were shown to be essential to catalytic activity, consistent with their proposed role in stabilization of apentacoordinate phosphate transition state resulting from in-line attack of the MEP phosphate on the-phosphate of CTP. In addition, Thr140, Arg109, Asp106, and Thr165 were all shown to play criticalroles in the binding and proper orientation of the MEP substrate.