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 biosynthetic enzyme in carrot cells, with phenylglyoxal yielded a chemically modified protein in which approximately two moles of the reagent were covalently attached to each subunit of the enzyme. Only NADH- but not NADPH-associated form of native 6-hydroxymellein synthase was inhibited by cerulenin; however, the NADPH-synthase complex lost the insensitivity by the chemical modification 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 catalytic 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 catalytic reaction of the native synthase-NADPH complex was appreciably decreased 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 amino acid residue, probably arginine, at the reaction center of 6-hydroxymellein synthase regulates several biochemical properties of this multifunctional enzyme.
Kurosaki
F
F
Faculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University, Sugitani, Toyama 930-0194, Japan. kurosaki@ms.toyama-mpu.ac.jp
Togashi
K
K
Arisawa
M
M
D000217:Acyltransferases / Q000037:antagonists & inhibitors / Q000737:chemistry*; D002384:Catalysis / Q000187:drug effects; D002569:Cerulenin / Q000494:pharmacology; D003065:Coenzyme A / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D007700:Kinetics; D008025:Ligases / Q000037:antagonists & inhibitors / Q000737:chemistry*; D008956:Models, Chemical; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors / Q000737:chemistry*; D009249:NADP / Q000737:chemistry*; D010088:Oxidoreductases / Q000037:antagonists & inhibitors / Q000737:chemistry*; D010658:Phenylglyoxal / Q000737:chemistry* / Q000494:pharmacology
D004791:Enzyme Inhibitors; D009097:Multienzyme Complexes; D002569:Cerulenin; D009249:NADP; D010088:Oxidoreductases; D000217:Acyltransferases; C070623:6-hydroxymellein synthetase; D008025:Ligases; D010658:Phenylglyoxal; D003065:Coenzyme A