Murine erythroid 5-aminolevulinate synthase: Adenosyl-binding site Lys221 modulates substrate binding and catalysis

摘要

5-Aminolevulinate synthase (ALAS) catalyzes the initial step of mammalian heme biosynthesis, the condensation between glycine and succinyl-CoA to produce CoA, CO"2, and 5-aminolevulinate. The crystal structure of Rhodobacter capsulatus ALAS indicates that the adenosyl moiety of succinyl-CoA is positioned in a mainly hydrophobic pocket, where the ribose group forms a putative hydrogen bond with Lys156. Loss-of-function mutations in the analogous lysine of human erythroid ALAS (ALAS2) cause X-linked sideroblastic anemia. To characterize the contribution of this residue toward catalysis, the equivalent lysine in murine ALAS2 was substituted with valine, eliminating the possibility of a hydrogen bond. The K221V substitution produced a 23-fold increase in the K"m^S^C^o^A and a 97% decrease in k"c"a"t/K"m^S^C^o^A. This reduction in the specificity constant does not stem from lower affinity toward succinyl-CoA, since the K"d^S^C^o^A of K221V is lower than that of wild-type ALAS. For both enzymes, the K"d^S^C^o^A value is significantly different from the K"m^S^C^o^A. That K221V has stronger binding affinity for succinyl-CoA was further deduced from substrate protection studies, as K221V achieved maximal protection at lower succinyl-CoA concentration than wild-type ALAS. Moreover, it is the CoA, rather than the succinyl moiety, that facilitates binding of succinyl-CoA to wild-type ALAS, as evident from identical K"d^S^C^o^A and K"d^C^o^A values. Transient kinetic analyses of the K221V-catalyzed reaction revealed that the mutation reduced the rates of quinonoid intermediate II formation and decay. Altogether, the results imply that the adenosyl-binding site Lys221 contributes to binding and orientation of succinyl-CoA for effective catalysis.