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

作者：Stojanovski Bosko M; Ferreira Gloria C^*

来源：FEBS Open Bio, 2015, 5(1): 824-831.

DOI：10.1016/j.fob.2015.09.009

• 5-Aminolevulinate synthase
• Pyridoxal phosphate
• Heme
• Enzymatic mechanism
• Sideroblastic anemia
• Porphyria

5-Aminolevulinate synthase (ALAS) catalyzes the initial step of mammalian heme biosynthesis, the condensation between glycine and succinyl-CoA to produce CoA, CO2, and 5-aminolevulinate. The crystal structure of Rhodobacter capsulatus ALAS indicates that the adenosyl moiety of succinylCoA 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(SCoA) and a 97% decrease in k(cat)/K-m(SCoA). This reduction in the specificity constant does not stem from lower affinity toward succinyl-CoA, since the K-d(SCoA) of K221V is lower than that of wild-type ALAS. For both enzymes, the K-d(SCoA) value is significantly different from the K-m(SCoA). 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(SCoA) and K-d(CoA) 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.

• 出版日期2015