A Bulky Hydrophobic Residue Is Not Required To Maintain the V‑Conformation of Enzyme-Bound Thiamin Diphosphate

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 belo...

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Bibliographic Details
Published in:Biochemistry (Easton) 2013-05, Vol.52 (18), p.3028-3030
Main Authors: Andrews, Forest H, Tom, Alan R, Gunderman, Peter R, Novak, Walter R. P, McLeish, Michael J
Format: Article
Language:English
Subjects:
Carboxy-Lyases - chemistry
Carboxy-Lyases - genetics
Carboxy-Lyases - metabolism
Molecular Conformation
Mutagenesis, Site-Directed
Thiamine Pyrophosphate - chemistry
Thiamine Pyrophosphate - metabolism
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Summary: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 k cat/K m 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.
ISSN:0006-2960
1520-4995
DOI:10.1021/bi400368j