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  • A bulky hydrophobic residue is not required to maintain the V-conformation of enzyme-bound thiamin diphosphate.

A bulky hydrophobic residue is not required to maintain the V-conformation of enzyme-bound thiamin diphosphate.

Biochemistry (2013-04-24)
Forest H Andrews, Alan R Tom, Peter R Gunderman, Walter R P Novak, Michael J McLeish
ABSTRACT

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.

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Product Description

Sigma-Aldrich
Thiamine pyrophosphate, ≥95%