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

FEBS Open Bio

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关键词: 5-Aminolevulinate synthase;    Pyridoxal phosphate;    Heme;    Enzymatic mechanism;    Sideroblastic anemia;    Porphyria;   

DOI  :  10.1016/j.fob.2015.09.009

来源: publisher

【 摘 要 】

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 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 theand a 97% decrease in . This reduction in the specificity constant does not stem from lower affinity toward succinyl‐CoA, since theof K221V is lower than that of wild‐type ALAS. For both enzymes, thevalue is significantly different from the . 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 identicalandvalues. 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.

【 授权许可】

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