Mutations at a glycine loop in aminolevulinate synthase affect pyridoxal phosphate cofactor binding and catalysis.

Abstract

5-Aminolevulinate synthase catalyzes the first step of the heme biosynthetic pathway in animals, fungi, and some bacteria. The enzyme belongs to a large family of enzymes that use pyridoxal 5'-phosphate as an essential cofactor. We previously analyzed the informational content contained in each residue of a conserved glycine loop, which we proposed to form part of the cofactor binding site [Gong, J., & Ferreira, G. C. (1995) Biochemistry 34, 1678-1685]. We found that Gly-142 and -144 contain high informational content, and we identified G144A, G144S, G144T, and G142C as functional mutants. Here, the catalytic parameters, cofactor affinities, and spectral and thermostability properties of these four glycine mutants are determined to examine the function of the glycine loop. In addition, computer models of the glycine loops from the wild-type and mutant enzymes were generated, using glycogen phosphorylase b as the structural template. G144A, G144S, G144T, and G142C displayed lower affinity than the wild-type enzyme for the cofactor, reflected in the 8.5-, 8-, 24.5-, and 15-fold increases, respectively, in the dissociation constant value for binding of the cofactor. While the turnover numbers for G144A, G144S, G144T, and G142C were 43%, 39%, 21%, and 6% of the wild-type value, respectively, the K(m) values for both substrates remained unchanged, with the exception of the G142C K(m)Gly, which showed a 4-fold increase. The UV-visible and CD spectra of Gly-144 mutants were similar to those of the wild type; however, the spectral properties of G142C suggest that this mutant binds the cofactor in a different mode at the active site. G144A, G144S, G144T, and G142C were also found to be less stable than the wild-type enzyme, with the thermotransition temperature, T1/2, determined to be 3.5, 3, 3.5, and 5 degrees C, respectively, lower than that of the wild-type enzyme. Collectively, computer modeling of the wild-type and mutant forms of the ALAS glycine loop and biochemical and spectroscopic characterization of G144A, G144S, G144T, and G142C strongly suggest that the conserved glycine loop in 5-aminolevulinate synthase is a pyridoxal 5'-phosphate cofactor binding motif.