title: Role of arginine 439 in substrate binding of 5-aminolevulinate synthase.
abstract: 5-Aminolevulinate synthase (EC 2.3.1.37) catalyzes the first reaction in the heme biosynthetic pathway in nonplant eukaryotes and some prokaryotes. Homology sequence modeling between 5-aminolevulinate synthase and some other alpha-family pyridoxal 5'-phosphate-dependent enzymes indicated that the residue corresponding to the Arg-439 of murine erythroid 5-aminolevulinate synthase is a conserved residue in this family of pyridoxal 5'-phosphate-dependent enzymes. Further, this conserved arginine residue in several enzymes, e.g., aspartate aminotransferase, for which the three-dimensional structure is known, has been shown to interact with the substrate carboxyl group. To test whether Arg-439 is involved in substrate binding in murine erythroid 5-aminolevulinate synthase, Arg-439 and Arg-433 of murine erythroid 5-aminolevulinate synthase were each replaced by Lys and Leu using site-directed mutagenesis. The R439K mutant retained 77% of the wild-type activity; its K(m) values for both substrates increased 9-13-fold, while the activity of R433K increased 2-fold and the K(m) values for both substrates remained unchanged. R439L had no measurable activity as determined using a standard 5-aminolevulinate synthase enzyme-coupled activity assay. In contrast, the kinetic parameters for R433L were comparable to those of the wild-type. Dissociation constants (Kd) for glycine increased 5-fold for R439K and at least 30-fold for R439L, while Kd values for glycine for both R433K and R433L mutants were similar to those of the wild-type. However, there was not much difference in methylamine binding among the mutants and the wild-type, excepting of a 10-fold increase in K(d)methylamine for R439L. R439K proved much less thermostable than the wild-type enzyme, with the thermotransition temperature, T1/2, determined to be 8.3 degrees C lower than that of the wild-type enzyme. In addition, in vivo complementation analysis demonstrated that in the active site of murine erythroid 5-aminolevulinate synthase, R439 is contributed from the same subunit as K313 (which is involved in the Schiff base linkage of the pyridoxal 5'-phosphate cofactor) and D279 (which interacts electrostatically with the ring nitrogen of the cofactor), while another subunit provides R149. Taken together, these findings suggest that Arg-439 plays an important role in substrate binding of murine erythroid 5-aminolevulinate synthase.
authors.affiliation: Department of Biochemistry and Molecular Biology, College of Medicine, H. Lee Moffitt Cancer Center and Research Institute, University of South Florida, Tampa 33612, USA.
authors.forename: D
authors.initials: D
authors.lastname: Tan
authors.forename: T
authors.initials: T
authors.lastname: Harrison
authors.forename: G A
authors.initials: GA
authors.lastname: Hunter
authors.forename: G C
authors.initials: GC
authors.lastname: Ferreira
mesh_terms: D000624:5-Aminolevulinate Synthetase / Q000097:blood / Q000235:genetics / Q000378:metabolism*; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D001120:Arginine / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D001224:Aspartic Acid / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D004789:Enzyme Activation / Q000235:genetics; D004795:Enzyme Stability / Q000235:genetics; D004912:Erythrocytes / Q000201:enzymology; D005998:Glycine / Q000378:metabolism; D007700:Kinetics; D008744:Methylamines / Q000378:metabolism; D051379:Mice; D016297:Mutagenesis, Site-Directed; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D008744:Methylamines; D001224:Aspartic Acid; D001120:Arginine; C027451:methylamine; D000624:5-Aminolevulinate Synthetase; D005998:Glycine