Isotopic Probes of the Enolase Reaction Mechanism

Abstract

Measurements are reported of initial and equilibrium rates of 2H, 3H, 14C, and 18O exchanges and of 3H secondary isotope effects in catalysis by muscle enolase. At equilibrium, with high substrate concentrations and at either pH 6.5 or 7.8, exchange of hydrogen on C-2 of 2-phosphoglycerate with water protons shows a prominent primary isotope rate effect. The estimated exchange rate of 1H is considerably greater than the rate of exchange of 18O between H18OH and the C-3 hydroxyl group. The 18O exchange is somewhat greater than 14C exchange between substrates at both pH values. In harmony with these equilibrium data, an incorporation of water protons into unreacted 2-phosphoglycerate is noted during net formation of phosphoenolpyruvate. No primary isotope effect on net formation of phosphoenolpyruvate is observed. The formation of 2-phosphoglycerate from phosphoenolpyruvate with 3H on C-3 shows an inverse secondary isotope rate effect. A proposed mechanism involves a central carbanion intermediate formed from 2-phosphoglycerate in a quasi-equilibrium step in accord with the isotope exchange data and formed from 2-phosphoenolpyruvate in a rate-limiting hydroxyl addition in accord with the inverse secondary isotope effect.