Role of asparagine 510 in the relative timing of substrate bond cleavages in the reaction catalyzed by choline oxidase.
The flavoprotein choline oxidase catalyzes the oxidation of choline to glycine betaine with transient formation of an aldehyde intermediate and molecular oxygen as final electron acceptor. The enzyme has been grouped in the glucose-methanol-choline oxidoreductase enzyme superfamily, which shares a highly conserved His-Asn catalytic pair in the active site. In this study, the conserved asparagine residue at position 510 in choline oxidase was replaced with alanine, aspartate, histidine, or leucine by site-directed mutagenesis, and the resulting mutant enzymes were purified and characterized in their biochemical and mechanistic properties. All of the substitutions resulted in low incorporation of FAD into the protein. The Asn510Asp enzyme was not catalytically active with choline and had 75% of the flavin associated noncovalently. The most notable changes in the catalytic parameters with respect to wild-type choline oxidase were seen in the Asn510Ala enzyme, with decreases of 4300-fold in the k(cat)/K(choline), 600-fold in the k(red), 660-fold in the k(cat), and 50-fold in the k(cat)/K(oxygen) values. Smaller, but nonetheless similar, changes were seen also in the Asn510His enzyme. Both the K(d) and K(m) values for choline changed < or = 7-fold. These data are consistent with Asn510 participating in both the reductive and oxidative half-reactions but having a minimal role in substrate binding. Substrate, solvent, and multiple kinetic isotope effects on the k(red) values indicated that the substitution of Asn510 with alanine, but not with histidine, resulted in a change from stepwise to concerted mechanisms for the cleavages of the OH and CH bonds of choline catalyzed by the enzyme.
Rungsrisuriyachai
Kunchala
K
Department of Chemistry, Georgia State University, Atlanta, Georgia 30302-4098, USA.
Gadda
Giovanni
G
D000429:Alcohol Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D001216:Asparagine; D055162:Biocatalysis; D020134:Catalytic Domain; D002417:Cattle; D005415:Flavins / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010084:Oxidation-Reduction; D013057:Spectrum Analysis
D005415:Flavins; D001216:Asparagine; D000429:Alcohol Oxidoreductases; C019888:choline oxidase