5-AMINOLEVULINATE SYNTHASE SCAFFOLD AND CONVERSION INTO A MORE ACTIVE ENZYME
J.-S. Zhang, G. A. Hunter and G. C. Ferreira
Department of Biochemistry and Molecular Biology, College of Medicine and H. Lee Moffitt Cancer Center and Research Institute, University of South Florida, Tampa, Florida 33612, USA.
5-Aminolevulinate synthase (ALAS), a pyridoxal 5’-phosphate-dependent enzyme, catalyzes the first step of the heme biosynthetic pathway in mammalian cells. This reaction entails the condensation of glycine with succinyl-coenzyme A to yield 5-aminolevulinate, carbon dioxide and CoA. The active site of the dimeric ALAS is located at the subunit interface with contribution of amino acids from the two subunits. Linking the two subunits into a single polypeptide chain dimer (2XALAS) yielded an enzyme with a ~7-fold greater turnover number than that of wild-type ALAS. Spectroscopic and kinetic properties of 2XALAS were investigated to explore the differences in the coenzyme structure and kinetic mechanism relative to those of wild-type ALAS that confer a more active enzyme. Transient kinetic analysis of the formation and decay of the quinonoid intermediate EQ2 indicated that, although their rates were similar in ALAS and 2XALAS, there was a greater accumulation of this intermediate in the 2XALAS-catalyzed reaction. Collectively, these results suggest that ketoenamine is the active form of the coenzyme and forms a more prominent coenzyme structure in 2XALAS than in ALAS at pH ~7.5.