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

Biochemistry. 2013 May 7;52(18):3028-30. doi: 10.1021/bi400368j. Epub 2013 Apr 23.

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Carboxy-Lyases / chemistry
  • Carboxy-Lyases / genetics
  • Carboxy-Lyases / metabolism*
  • Molecular Conformation
  • Mutagenesis, Site-Directed
  • Thiamine Pyrophosphate / chemistry*
  • Thiamine Pyrophosphate / metabolism

Substances

  • Carboxy-Lyases
  • benzoylformate decarboxylase
  • Thiamine Pyrophosphate