Rescuing of the hydride transfer reaction in the Glu312Asp variant of choline oxidase by a substrate analogue

Abstract

In the active site of choline oxidase, Glu312 participates in binding the trimethylammonium group of choline, thereby positioning the alcohol substrate properly for efficient hydride transfer to the enzyme-bound flavin. Previous studies have shown that substitution of Glu312 with aspartate results in a perturbed mechanism of hydride transfer, with a 260-fold decrease in the rate associated with the mutation. Here, the reaction of alcohol oxidation catalyzed by the Glu312Asp enzyme has been investigated with 3-hydroxypropyl-trimethylamine (3-HPTA), a choline analogue with an extra methylene, as substrate. The results of the kinetic investigation using steady state and rapid reaction approaches showed that the impaired ability of the Glu312Asp enzyme to catalyze a hydride transfer reaction can be effectively, but not completely, rescued in the presence of an extra methylene group on the substrate that compensates for the equivalent shortening of the side chain on residue 312. This observation is consistent with choline oxidase having evolved to optimally catalyze the oxidation of choline.