THE MECHANISM OF RABBIT MUSCLE ENOLASE - IDENTIFICATION OF THE RATE-LIMITING STEPS AND THE SITE OF LI+ INHIBITION

Steady-state and non-steady-state techniques have been used to identif y the rate-limiting steps for beta beta enolase (rabbit muscle enolase ), at pH 7.1, with Mn2+ as the required cation. A minimum mechanism fo r enolase includes eight steps, [GRAPHICS] where S is phosphoglycerate , P is phosphoenolpyruvate (PEP), I is the carbanion intermediate, M i s Me(2+), and EM is the holoenolase (i.e., the first Me(2+) is bound). Asterisks represent a different conformation of the quaternary comple xes. At pH 7.1, the primary kinetic isotope effect = 1, and k(cat) dec reases as solvent viscosity increases. The changes in protein fluoresc ence that occur upon substrate binding and product release [EMSM rever sible arrow (EMSM) and (EMPM)* reversible arrow EMPM] were followed b y stopped-flow fluorimetry; the viscosity dependence of the observed r ates was also determined. The data support the following mechanism. Pr oduct formation is fast and precedes the slow steps of the reaction, c onsistent with the observation of a presteady-state burst of PEP. The rate-limiting steps are k(+6), the conformational change associated wi th product release, and k(+8), the dissociation of PEP. Li+ inhibits t he activity of enolase by increasing k(+6) and k(-s), thus decreasing the steady-state concentration of (EMSM). (C) 1996 Academic Press, In c.