type	Journal Article
authors	Zhang, J.; Ferreira, G. C.
title	Transient-state kinetic investigation of 5-aminolevulinate synthase reaction mechanism
journal	J Biol Chem
Activity	2.3.1.37
ui	Tskiasr
year	(2002)
volume	20
pages	20
abstract	5-Aminolevulinate synthase (ALAS), a pyridoxal 5'-phosphate (PLP)- dependent enzyme, catalyzes the first, and regulatory, step of the heme biosynthetic pathway in non-plant 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 PLP-glycine aldimine, followed by the abstraction of the glycine pro-R proton from the external aldimine. Significantly, the rate associated with this third step (k3 = 0.002 s- 1) is consistent with the rate determined for the ALAS-catalyzed removal of tritium from [2-3H2]-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. J. Biol. Chem. 1999, 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.
last changed	2002/11/04 17:52