STUDIES ON PIG MUSCLE ALDOSE REDUCTASE - KINETIC MECHANISM AND EVIDENCE FOR A SLOW CONFORMATIONAL CHANGE UPON COENZYME BINDING

Steady state kinetic analysis at pH 7.0 of the reduction of DL-glycera ldehyde by pig muscle aldose reductase showed that the enzyme follows a sequential ordered mechanism with NADPH binding first. However, the "off constant" for NADP+ in the forward direction was 1 order of magni tude less than the k(cat). Analysis of this anomaly by pre-steady stat e kinetics using stopped-flow fluorescence spectroscopy showed that th is could be accounted for by isomerization of the enzyme-NADP+ complex and that the rate of isomerization is the rate-limiting step. The rat e constant for this step was of the same order of magnitude as the k(c at) for the forward reaction. Fluorescence emission spectra of free an d NADP(H)-bound enzyme suggested a conformational change upon binding of coenzyme. In the reverse direction (oxidation of glycerol) pre-stea dy state and steady state kinetic analyses were consistent with the ra te-limiting step occurring before isomerization of the enzyme-NADPH co mplex. We conclude, therefore, that during the kinetic mechanism of th e reduction of aldehydes by aldose reductase, a slow (kinetically dete ctable) conformational change in the enzyme occurs upon coenzyme bindi ng. Since NADPH and NADP+ bind to the enzyme very tightly, this has im plications for the targeting and binding of drugs that are aldose redu ctase inhibitors.