Transient State Kinetic Investigation of 5-Aminolevulinate Synthase Reaction Mechanism

5-Aminolevulinate synthase (ALAS), a pyridoxal 5′-phosphate-dependent enzyme, catalyzes the first, and regulatory, step of the heme biosynthetic pathway in nonplant eukaryotes and some bacteria. 5-Aminolevulinate synthase is a dimeric protein having an ordered kinetic mechanism with glycine binding before succinyl-CoA and with aminolevulinate release after CoA and carbon dioxide. Rapid scanning stopped-flow absorption spectrophotometry in conjunction with multiple turnover chemical quenched-flow kinetic analyses and a newly developed CoA detection method were used to examine the ALAS catalytic reaction and identify the rate-determining step. The reaction of glycine with ALAS follows a three-step kinetic process, ascribed to the formation of the Michaelis complex and the pyridoxal 5′-phosphate-glycine aldimine, followed by the abstraction of the glycine pro -R proton from the external aldimine. Significantly, the rate associated with this third step ( k 3 = 0.002 s −1 ) is consistent with the rate determined for the ALAS-catalyzed removal of tritium from [2- 3 H 2 ]glycine. Succinyl-CoA and acetoacetyl-CoA increased the rate of glycine proton removal ∼250,000- and 10-fold, respectively, supporting our previous proposal that the physiological substrate, succinyl-CoA, promotes a protein conformational change, which accelerates the conversion of the external aldimine into the initial quinonoid intermediate (Hunter, G. A., and Ferreira, G. C. (1999) J. Biol. Chem. 274, 12222–12228). Rapid scanning stopped-flow and quenched-flow kinetic analyses of the ALAS reaction under single turnover conditions lend evidence for two quinonoid reaction intermediates and a model of the ALAS kinetic mechanism in which product release is at least the partially rate-limiting step. Finally, the carbonyl and carboxylate groups of 5-aminolevulinate play a major protein-interacting role by inducing a conformational change in ALAS and, thus, possibly modulating product release.