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 approximately 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.