On the contribution of the positively charged headgroup of choline to substrate binding and catalysis in the reaction catalyzed by choline oxidase

Recent kinetic studies established that the positive charge on the trimethylammonium group of choline plays an important role in substrate binding and specificity in the reaction catalyzed by choline oxidase. In the present study, pH and solvent viscosity effects with the isosteric analogue of choline 3,3-dimethyl-butan-1-ol have been used to further dissect the contribution of the substrate positive charge to substrate binding and catalysis in the reaction catalyzed by choline oxidase. Both the k(cat) and k(cat)/K-m values with 3,3-dimethylbutan-1-ol increased to limiting values that were similar to 3- and similar to 400-times lower than those observed with choline, defining pK(a) values that were similar to the thermodynamic pK(a) value of similar to 7.5 previously determined. No effects of increased solvent viscosity were observed on the k(cat) and k(cat)/K-m values with the substrate analogue at pH 8, suggesting that the chemical step of substrate oxidation is fully rate-limiting for the overall turnover and the reductive half-reaction in which the alcohol substrate is oxidized to the aldehyde. The k(cat)/ Km value for oxygen determined with the substrate analogue was pH-independent in the pH range from 6 to 10, with an average value that was similar to 75-times lower than that previously determined with choline as substrate. These data are consistent with the positive charge headgroup of choline playing important roles for substrate binding and flavin oxidation, with minimal contribution to substrate oxidation. (c) 2006 Elsevier Inc. All rights reserved.