Pre-steady-state reaction of 5-aminolevulinate synthase. Evidence for a rate-determining product release.
5-Aminolevulinate synthase (ALAS) is the first enzyme of the heme biosynthetic pathway in non-plant eukaryotes and the alpha-subclass of purple bacteria. The pyridoxal 5'-phosphate cofactor at the active site undergoes changes in absorptive properties during substrate binding and catalysis that have allowed us to study the kinetics of these reactions spectroscopically. Rapid scanning stopped-flow experiments of murine erythroid 5-aminolevulinate synthase demonstrate that reaction with glycine plus succinyl-CoA results in a pre-steady-state burst of quinonoid intermediate formation. Thus, a step following binding of substrates and initial quinonoid intermediate formation is rate-determining. The steady-state spectrum of the enzyme is similar to that formed in the presence of 5-aminolevulinate, suggesting that release of this product limits the overall rate. Reaction of either glycine or 5-aminolevulinate with ALAS is slow (kf = 0.15 s-1) and approximates kcat. The rate constant for reaction with glycine is increased at least 90-fold in the presence of succinyl-CoA and most likely represents a slow conformational change of the enzyme that is accelerated by succinyl-CoA. The slow rate of reaction of 5-aminolevulinate with ALAS is 5-aminolevulinate-independent, suggesting that it also represents a slow isomerization of the enzyme. Reaction of succinyl-CoA with the enzyme-glycine complex to form a quinonoid intermediate is a biphasic process and may be irreversible. Taken together, the data suggest that turnover is limited by release of 5-aminolevulinate or a conformational change associated with 5-aminolevulinate release.
Hunter
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GA
Department of Biochemistry and Molecular Biology, College of Medicine, University of South Florida, Tampa, Florida 33612, USA. gferreir@com1.med.usf.edu
Ferreira
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GC
D000624:5-Aminolevulinate Synthetase / Q000378:metabolism*; D000818:Animals; D004912:Erythrocytes / Q000201:enzymology; D005998:Glycine / Q000378:metabolism; D007700:Kinetics; D051379:Mice; D011809:Quinones / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D013057:Spectrum Analysis; D013379:Substrate Specificity
D011809:Quinones; D011994:Recombinant Proteins; D000624:5-Aminolevulinate Synthetase; D005998:Glycine