ROLE OF ARGININE-439 IN SUBSTRATE-BINDING OF 5-AMINOLEVULINATE SYNTHASE

5-Aminolevulinate synthase (EC 2.3.1.37) catalyzes the first reaction in the heme biosynthetic pathway in nonplant eukaryotes and some proka ryotes. Homology sequence modeling between 5-aminolevulinate synthase and some other alpha-family pyridoxal 5'-phosphate-dependent enzymes i ndicated that the residue corresponding to the Arg-439 of murine eryth roid 5-aminolevulinate synthase is a conserved residue in this family of pyridoxal 5'-phosphate-dependent enzymes. Further, this conserved a rginine 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 s ynthase, Arg-439 and Arg-433 of murine erythroid 5-aminolevulinate syn thase were each replaced by Lys and Leu using site-directed mutagenesi s. The R439K mutant retained 77% of the wild-type activity; its K-m va lues for both substrates increased 9-13-fold, while the activity of R4 33K increased 2-fold and the K-m values for both substrates remained u nchanged, R439L had no measurable activity as determined using a stand ard 5-aminolevulinate synthase enzyme-coupled activity assay, in contr ast, the kinetic parameters for R433L were comparable to those of the wild-type. Dissociation constants (K-d) for glycine increased 5-fold f or R439K and at least 30-fold for R439L, while K-d values for glycine for both R433K and R433L mutants were similar to those of the wild-typ e, 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, T-1/2, determ ined to be 8.3 degrees C lower than that of the wild-type enzyme, In a ddition, in vivo complementation analysis demonstrated that in the act ive site of murine erythroid 5-aminolevulinate synthase, R439 is contr ibuted from the same subunit as K313 (which is involved in the Schiff base linkage of the pyridoxal 5'-phosphate cofactor) and D279 (which i nteracts electrostatically with the ring nitrogen of the cofactor), wh ile another subunit provides R149. Taken together, these findings sugg est that Arg-439 plays an important role in substrate binding of murin e erythroid 5-aminolevulinate synthase.