Structural and Kinetic Analysis of Catalysis by a Thiamin Diphosphate-Dependent Enzyme, Benzoylformate Decarboxylase

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• 作者：Elena S. Polovnikova ; Michael J. McLeish ; Eduard A. Sergienko ; John T. Burgner ; Natalie L. Anderson ; Asim K. Bera ; Frank Jordan ; George L. Kenyon ; and Miriam S. Hasson
• 刊名：Biochemistry
• 出版年：2003
• 出版时间：February 25, 2003
• 年：2003
• 卷：42
• 期：7
• 页码：1820 - 1830
• 全文大小：595K
• 年卷期：v.42,no.7(February 25, 2003)
• ISSN：1520-4995

文摘

Benzoylformate decarboxylase is a member of the family of enzymes that are dependent onthe cofactor thiamin diphosphate. A structure of this enzyme binding (R)-mandelate, a competitive inhibitor,suggests that at least two hydrogen bonds are formed between the substrate, benzoylformate, and activesite side chains. The first is between the carboxylate group of benzoylformate and the hydroxyl group ofS26, and the second is between carbonyl group of the substrate and an imidazole nitrogen of H70. Steady-state kinetic studies indicate that the catalytic parameters are strongly affected in three active site mutants,S26A, H70A, and H281A. The K_m of S26A was increased most dramatically, 25-fold more than that ofthe wild-type enzyme, while the K_i of (R)-mandelate was increased 100-fold, suggesting that the serinehydroxyl is important for substrate binding. The k_cat of H70A is reduced more than 3 orders of magnitude,strongly implicating this residue in catalysis, and H281 showed significant, but smaller magnitude, effectson both K_m and k_cat. Stopped-flow experiments using an alternative substrate, p-nitrobenzoylformate, leadto kinetic resolution of the fate of key thiamin diphosphate-bound intermediates. Together, the experimentalresults suggest the following roles for residues in the active site. The residue H70 is important for theprotonation of the 2--mandelyl-ThDP intermediate, thereby assisting in decarboxylation, and for thedeprotonation of the 2--hydroxybenzyl-ThDP intermediate, aiding product release. H281 is involved inprotonation of the enamine. Surprisingly, S26 appears to be involved not only in substrate binding butalso in other steps of the reaction.