title: Murine erythroid 5-aminolevulinate synthase: Adenosyl-binding site Lys221 modulates substrate binding and catalysis.
abstract: 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 the [Formula: see text] and a 97% decrease in [Formula: see text]. This reduction in the specificity constant does not stem from lower affinity toward succinyl-CoA, since the [Formula: see text] of K221V is lower than that of wild-type ALAS. For both enzymes, the [Formula: see text] value is significantly different from the [Formula: see text]. 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 [Formula: see text] and [Formula: see text] 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.
authors.affiliation: Department of Molecular Medicine, Morsani College of Medicine, University of South Florida, Tampa, FL 33612, United States.
authors.forename: Bosko M
authors.initials: BM
authors.lastname: Stojanovski
authors.affiliation: Department of Molecular Medicine, Morsani College of Medicine, University of South Florida, Tampa, FL 33612, United States ; Department of Chemistry, University of South Florida, Tampa, FL 33612, United States.
authors.forename: Gloria C
authors.initials: GC
authors.lastname: Ferreira
keywords: 5-Aminolevulinate synthase; ALA, 5-aminolevulinic acid; ALAS2, erythroid specific isoform of 5-aminolevulinate synthase; Enzymatic mechanism; Heme; PLP, pyridoxal 5′-phosphate; Porphyria; Pyridoxal phosphate; SDS–PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; Sideroblastic anemia; XLPP, X-linked protoporphyria; XLSA, X-linked sideroblastic anemia; mALAS2, erythroid specific isoform of murine 5-aminolevulinate synthase