KINETIC ISOTOPE EFFECTS ON SUBSTRATE ASSOCIATION - REACTIONS OF PHOSPHOENOLPYRUVATE WITH PHOSPHOENOLPYRUVATE CARBOXYLASE AND PYRUVATE-KINASE

Kinetic isotope effects on association have been measured using the re mote label methodology developed by O'Leary and Marlier (1979). The is otope effect on V/K-A for the first substrate in an obligatorily order ed mechanism is an isotope effect on its second-order rate constant fo r association with the enzyme. With phosphoenolpyruvate carboxylase th e (18)(V/K-PEP) when the bridging O is labeled decreases from 1.0056 /- 0.0007 to 0.9943 +/- 0.0002 as the concentration of bicarbonate, th e second substrate, increases from 2 to 200 mM. With pyruvate kinase t he (18)(V/K-PEP) decreases from 1.0024 +/- 0.0014 to 0.9928 +/- 0.0027 as the concentration of ADP increases from 1.5 to 30 mM. These invers e kinetic isotope effects are best understood as arising from an isoto pe effect on the rate constant for forming the Michaelis complex of en zyme and substrate. The inverse value suggests that the bridging oxyge n is in a vibrationally stiffer environment in the transition state fo r the association reaction.