Asn-150 of Murine Erythroid 5-Aminolevulinate Synthase Modulates the Catalytic Balance between the Rates of the Reversible Reaction.

摘要: 5-Aminolevulinate synthase (ALAS) catalyzes the first step in mammalian heme biosynthesis, pyridoxal 5'-phosphate (PLP)-dependent and reversible reaction between glycine succinyl-CoA to generate CoA, CO2, 5-aminolevulinate (ALA). Apart from coordinating positioning of succinyl-CoA, Rhodobacter capsulatus ALAS Asn-85 has a proposed role regulating opening an active site channel. Here, we constructed library murine erythroid variants with substitutions at position occupied by analogous bacterial asparagine, screened for function, characterized catalytic properties N150H N150F variants. Quinonoid intermediate formation occurred significantly reduced rate either N150H- or N150F-catalyzed condensation during single turnover. The introduced mutations caused modifications such that resulting tipped balance forward- reverse-catalyzed reactions. Although wild-type conversion ALA into quinonoid 6.3-fold slower than same variant forward mere 1.2-fold faster reverse reaction, reverses values 1.7-fold reaction. We conclude evolutionary selection Asn-150 was significant optimizing enzymatic expense reverse, thus ensuring is predominantly available biosynthesis.