Structural and kinetic studies on the Ser101Ala variant of choline oxidase: Catalysis by compromise

Journal Article · Wed Sep 15 00:00:00 EDT 2010 · Archives of Biochemistry and Biophysics

DOI:https://doi.org/10.1016/j.abb.2010.06.014· OSTI ID:993049

Finnegan, S; Orville, A; Yuan, H; Wang, Y -F; Weber, I T; Gadda, G

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.

• Research Organization: Brookhaven National Lab. (BNL), Upton, NY (United States)
• Sponsoring Organization: DOE - Office Of Science
• DOE Contract Number: DE-AC02-98CH10886
• OSTI ID: 993049
• Report Number(s): BNL-93985-2010-JA; ABBIA4; R&D Project: BO-070; KP1605010; TRN: US201023%%198
• Journal Information: Archives of Biochemistry and Biophysics, Vol. 501, Issue 2; ISSN 0003-9861
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
99 GENERAL AND MISCELLANEOUS//MATHEMATICS, COMPUTING, AND INFORMATION SCIENCE
ALANINES
ALCOHOLS
ALDEHYDES
CARBOXYLIC ACIDS
CATALYSIS
CHOLINE
CONFIGURATION
ENZYMES
ISOALLOXAZINES
KINETICS
MUTAGENESIS
MUTANTS
MUTATIONS
OPTIMIZATION
OXIDASES
OXIDATION
OXYGEN
RESIDUES
SUBSTRATES
Oxidase
Flavoprotein
Kinetics
Mechanism
Reductive half-reaction
Choline