Transient kinetic studies support refinements to the chemical and kinetic mechanisms of aminolevulinate synthase

Abstract

5-Aminolevulinate synthase catalyzes the pyridoxal 5′-phosphate- dependent condensation of glycine and succinyl-CoA to produce carbon dioxide, CoA, and 5-aminolevulinate, in a reaction cycle involving the mechanistically unusual successive cleavage of two amino acid substrate α-carbon bonds. Single and multiple turnover rapid scanning stopped-flow experiments have been conducted from pH 6.8-9.2 and 5-35 °C, and the results, interpreted within the framework of the recently solved crystal structures, allow refined characterization of the central kinetic and chemical steps of the reaction cycle. Quinonoid intermediate formation occurs with an apparent pKa of 7.7 ± 0.1, which is assigned to His-207 acid-catalyzed decarboxylation of the α-amino-β-ketoadipate intermediate to form an enol that is in rapid equilibrium with the 5-aminolevulinate-bound quinonoid species. Quinonoid intermediate decay occurs in two kinetic steps, the first of which is acid-catalyzed with a pKa of 8.1 ± 0.1, and is assigned to protonation of the enol by Lys-313 to generate the product-bound external aldimine. The second step of quinonoid decay defines kcat and is relatively pH-independent and is assigned to opening of the active site loop to allow ALA dissociation. The data support important refinements to both the chemical and kinetic mechanisms and indicate that 5-aminolevulinate synthase operates under the stereoelectronic control predicted by Dunathan's hypothesis. © 2007 by The American Society for Biochemistry and Molecular Biology, Inc.