Structural and kinetic studies on the Ser101Ala variant of choline oxidase: catalysis by compromise.
The oxidation of choline catalyzed by choline oxidase includes two reductive half-reactions where FAD is reduced by the alcohol substrate and by an aldehyde intermediate transiently formed in the reaction. Each reductive half-reaction is followed by an oxidative half-reaction where the reduced flavin is oxidized by oxygen. Here, we have used mutagenesis to prepare the Ser101Ala mutant of choline oxidase and have investigated the impact of this mutation on the structural and kinetic properties of the enzyme. The crystallographic structure of the Ser101Ala enzyme indicates that the only differences between the mutant and wild-type enzymes are the lack of a hydroxyl group on residue 101 and a more planar configuration of the flavin in the mutant enzyme. Kinetics established that replacement of Ser101 with alanine yields a mutant enzyme with increased efficiencies in the oxidative half-reactions and decreased efficiencies in the reductive half-reactions. This is accompanied by a significant decrease in the overall rate of turnover with choline. Thus, this mutation has revealed the importance of a specific residue for the optimization of the overall turnover of choline oxidase, which requires fine-tuning of four consecutive half-reactions for the conversion of an alcohol to a carboxylic acid.
Finnegan
Steffan
S
Department of Chemistry, Georgia State University, Atlanta, GA 30302-4098, USA.
Yuan
Hongling
H
Wang
Yuan-Fang
YF
Orville
Allen M
AM
Weber
Irene T
IT
Gadda
Giovanni
G
D000429:Alcohol Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019943:Amino Acid Substitution; D001173:Arthrobacter / Q000201:enzymology / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002794:Choline / Q000378:metabolism; D018360:Crystallography, X-Ray; D014644:Genetic Variation; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D055672:Static Electricity
D001426:Bacterial Proteins; D050505:Mutant Proteins; D011994:Recombinant Proteins; D000429:Alcohol Oxidoreductases; C019888:choline oxidase; D002794:Choline