title: 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 beta beta enolase (rabbit muscle enolase), at pH 7.1, with Mn2+ as the required cation. A minimum mechanism for enolase includes eight steps, [see text] 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, and kappa(cat) decreases as solvent viscosity increases. The changes in protein fluorescence that occur upon substrate binding and product release [EMSM <-> (EMSM)* and (EMPM)* <-> 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 are kappa(+6) the conformational change associated with product release, and kappa(+8) the dissociation of PEP. Li+ inhibits the activity of enolase by increasing kappa(+6) and kappa(-3), thus decreasing the steady-state concentration of (EMSM)*.
authors.affiliation: Enzyme Research Group, Department of Chemistry and Biochemistry, Concordia University, Montreal, Quebec, Canada.
authors.forename: M J
authors.initials: MJ
authors.lastname: Kornblatt
mesh_terms: D000818:Animals; D001665:Binding Sites; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008094:Lithium / Q000494:pharmacology*; D008433:Mathematics; D018482:Muscle, Skeletal / Q000201:enzymology*; D010751:Phosphopyruvate Hydratase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D011817:Rabbits; D012984:Software
chemical_list: D008094:Lithium; D010751:Phosphopyruvate Hydratase