A Bulky Hydrophobic Residue Is Not Required To Maintain the V-Conformation of Enzyme-Bound Thiamin Diphosphate
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- 作者：Forest H. Andrews ; Alan R. Tom ; Peter R. Gunderman ; Walter R. P. Novak ; Michael J. McLeish
- 刊名：Biochemistry
- 出版年：2013
- 出版时间：May 7, 2013
- 年：2013
- 卷：52
- 期：18
- 页码：3028-3030
- 全文大小：181K
- 年卷期：v.52,no.18(May 7, 2013)
- ISSN：1520-4995
文摘
It is widely accepted that, in thiamin diphosphate (ThDP)-dependent enzymes, much of the rate acceleration is provided by the cofactor. Inter alia, the reactive conformation of ThDP, known as the V-conformation, has been attributed to the presence of a bulky hydrophobic residue located directly below the cofactor. Here we report the use of site-saturation mutagenesis to generate variants of this residue (Leu403) in benzoylformate decarboxylase. The observed 3 orders of magnitude range in kcat/Km values suggested that conformational changes in the cofactor could be influencing catalysis. However, X-ray structures of several variants were determined, and there was remarkably little change in ThDP conformation. Rather, it seemed that, once the V-conformation was attained, residue size and hydrophobicity were more important for enzyme activity.