title: A bulky hydrophobic residue is not required to maintain the V-conformation of enzyme-bound thiamin diphosphate.
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.
authors.affiliation: Department of Chemistry and Chemical Biology, Indiana University-Purdue University Indianapolis, Indianapolis, IN 46202, USA.
authors.forename: Forest H
authors.initials: FH
authors.lastname: Andrews
authors.forename: Alan R
authors.initials: AR
authors.lastname: Tom
authors.forename: Peter R
authors.initials: PR
authors.lastname: Gunderman
authors.forename: Walter R P
authors.initials: WR
authors.lastname: Novak
authors.forename: Michael J
authors.initials: MJ
authors.lastname: McLeish
mesh_terms: D002262:Carboxy-Lyases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008968:Molecular Conformation; D016297:Mutagenesis, Site-Directed; D013835:Thiamine Pyrophosphate / Q000737:chemistry* / Q000378:metabolism
chemical_list: D002262:Carboxy-Lyases; C019778:benzoylformate decarboxylase; D013835:Thiamine Pyrophosphate