Asn-150 of Murine Erythroid 5-Aminolevulinate Synthase Modulates the Catalytic Balance between the Rates of the Reversible Reaction.
5-Aminolevulinate synthase (ALAS) catalyzes the first step in mammalian heme biosynthesis, the pyridoxal 5'-phosphate (PLP)-dependent and reversible reaction between glycine and succinyl-CoA to generate CoA, CO2, and 5-aminolevulinate (ALA). Apart from coordinating the positioning of succinyl-CoA, Rhodobacter capsulatus ALAS Asn-85 has a proposed role in regulating the opening of an active site channel. Here, we constructed a library of murine erythroid ALAS variants with substitutions at the position occupied by the analogous bacterial asparagine, screened for ALAS function, and characterized the catalytic properties of the N150H and N150F variants. Quinonoid intermediate formation occurred with a significantly reduced rate for either the N150H- or N150F-catalyzed condensation of glycine with succinyl-CoA during a single turnover. The introduced mutations caused modifications in the ALAS active site such that the resulting variants tipped the balance between the forward- and reverse-catalyzed reactions. Although wild-type ALAS catalyzes the conversion of ALA into the quinonoid intermediate at a rate 6.3-fold slower than the formation of the same quinonoid intermediate from glycine and succinyl-CoA, the N150F variant catalyzes the forward reaction at a mere 1.2-fold faster rate than that of the reverse reaction, and the N150H variant reverses the rate values with a 1.7-fold faster rate for the reverse reaction than that for the forward reaction. We conclude that the evolutionary selection of Asn-150 was significant for optimizing the forward enzymatic reaction at the expense of the reverse, thus ensuring that ALA is predominantly available for heme biosynthesis.
From the Department of Molecular Medicine, Morsani College of Medicine, and.
Bosko M
BM
Stojanovski
From the Department of Molecular Medicine, Morsani College of Medicine, and the Department of Chemistry, University of South Florida, Tampa, Florida 33612 gferreir@health.usf.edu.
Gloria C
GC
Ferreira
D000624:5-Aminolevulinate Synthetase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D000214:Acyl Coenzyme A; D019943:Amino Acid Substitution; D000622:Aminolevulinic Acid / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D001216:Asparagine / Q000235:genetics / Q000378:metabolism; D055162:Biocatalysis; D020134:Catalytic Domain; D041905:Erythroid Cells / Q000737:chemistry / Q000201:enzymology*; D005998:Glycine / Q000378:metabolism; D007700:Kinetics; D051379:Mice; D013379:Substrate Specificity
D000214:Acyl Coenzyme A; D001216:Asparagine; D000622:Aminolevulinic Acid; C012046:succinyl-coenzyme A; D000624:5-Aminolevulinate Synthetase; D005998:Glycine
5-aminolevulinate synthase; enzyme mechanism; enzyme mutation; heme; porphyria; porphyrin; pyridoxal phosphate; reaction selectivity; sideroblastic anemia; substrate specificity