Water-d2 effect on catalysis by triose phosphate isomerase requires isotope exchange on the enzyme

Abstract

The present work shows the value of measuring the rate of onset or loss of a D/sub 2/O effect to distinguish between enzyme-bound protons and protons of the medium as the cause of the effect. This alternative was examined in the important studies of Bender on deacylation of chymotrypsin and papain in which the isotope effect in D/sub 2/O was lost within 10 s of dilution into H/sub 2/O and therefore was concluded to result from D/sub 2/O rather than from a deuterium-substituted enzyme. However, 10 s is a long interval for all but the hydrogen-bonded backbone hydrogens. With the ususal rapid mixing device this interval can be shortened by a least 10/sup 3/, putting it in the range of many group ionizations. The results suggest that H/D exchange on the enzyme, occurring more slowly than catalysis, is required for the isotope effect to be seen. Because the isotope effect implies that a proton transfer within the enzyme occurs at some step of the reaction and because exchange is much slower than catalysis, the transfer must be reversed without exchange with the medium during product formation. It is proposed that a conformational change of E-G3P prior to the enolization ismore » the step requiring such a proton transfer.« less