On the Role of Histidine 351 in the Reaction of Alcohol Oxidation Catalyzed by Choline Oxidase

Abstract

Choline oxidase catalyzes the four-electron, flavin-linked oxidation of choline to glycine betaine with transient formation of an enzyme-bound aldehyde intermediate. The recent determination of the crystal structure of choline oxidase to a resolution of 1.86 Å established the presence of two histidine residues in the active site, which may participate in catalysis. His466 was the subject of a previous study [Ghanem, M., and Gadda, G. (2005) Biochemistry 44, 893−904]. In this study, His351 was replaced with alanine using site-directed mutagenesis, and the resulting mutant enzyme was purified and characterized in its mechanistic properties. The results presented establish that His351 contributes to substrate binding and positioning and stabilizes the transition state for the hydride transfer reaction to the flavin, as suggested by anaerobic substrate reduction stopped-flow data. Furthermore, His351 contributes to the overall polarity of the active site by modulating the pKa of the group that deprotonates choline to the alkoxide species, as indicated by pH profiles of the steady-state kinetic parameters with the substrate or a competitive inhibitor. Surprisingly, His351 is not involved in the activation of the reduced flavin for reaction with oxygen. The latter observation, along with previous mutagenesis data on His466, allow us to conclude that choline oxidase must necessarily utilize a strategy for oxygen reduction different from that established for glucose oxidase, where other authors showed that the catalytic effect almost entirely arises from a protonated histidine residue