Regulation of catalytic activity of a multifunctional polyketide biosynthetic enzyme, 6-hydroxymellein synthase, by interaction between NADPH and phenylglyoxal-sensitive amino acid residue at the reaction center

Treatment of 6-hydroxymellein synthase, a multifunctional polyketide biosyn thetic enzyme in carrot cells, with phenylglyoxal yielded a chemically modi fied protein in which approximately two moles of the reagent were covalentl y attached to each subunit of the enzyme. Only NADH- but not NADPH-associat ed form of native 6-hydroxymellein synthase was inhibited by cerulenin; how ever, the NADPH-synthase complex lost the insensitivity by the chemical mod ification of the enzyme protein with phenylglyoxal. Appreciable differences in K-m values observed between the NADPH- and NADH-associated enzymes were greatly reduced by the treatment with phenylglyoxal. Although the catalyti c activity of the NADPH-associated synthase was enhanced by the addition of free CoA. the compound exhibited a significant inhibitory activity to the phenylglyoxal-modified enzyme, A marked deuterium isotope effect in the cat alytic reaction of the native synthase-NADPH complex was appreciably decrea sed in the chemically modified enzyme. These results strongly suggest that an electrostatic interaction between the phosphate group attached to the 2 ' -position of adenosyl moiety of NADPH and the phenylglyoxal-sensitive ami no acid residue, probably arginine, at the reaction center of 6-hydroxymell ein synthase regulates several biochemical properties of this multifunction al enzyme. (C) 2001 Elsevier Science BN. All rights reserved.