The Mechanism of Rabbit Muscle Enolase: Identification of the Rate-Limiting Steps and the Site of Li+Inhibition

Abstract

Steady-state and non-steady-state techniques have been used to identify the rate-limiting steps for ββ enolase (rabbit muscle enolase), at pH 7.1, with Mn2+as the required cation. A minimum mechanism for enolase includes eight steps,[formula]where S is phosphoglycerate, P is phosphoenolpyruvate (PEP), I is the carbanion intermediate, M is Me2+, and EM is the holoenolase (i.e., the first Me2+is bound). Asterisks represent a different conformation of the quaternary complexes. At pH 7.1, the primary kinetic isotope effect = 1, andkcatdecreases as solvent viscosity increases. The changes in protein fluorescence that occur upon substrate binding and product release [EMSM[formula](EMSM)* and (EMPM)*[formula]EMPM] were followed by stopped-flow fluorimetry; the viscosity dependence of the observed rates was also determined. The data support the following mechanism. Product formation is fast and precedes the slow steps of the reaction, consistent with the observation of a pre-steady-state burst of PEP. The rate-limiting steps arek+6, the conformational change associated with product release, andk+8, the dissociation of PEP. Li+inhibits the activity of enolase by increasingk+6andk−3, thus decreasing the steady-state concentration of (EMSM)*.