title: Chemical modification of an arginine residue in aldose reductase is enhanced by coenzyme binding: further evidence for conformational change during the reaction mechanism.
abstract: Chemical modification of pig muscle aldose reductase (ALR2) with the arginine specific reagent phenylglyoxal resulted in the inactivation of the enzyme. This inactivation exhibited pseudo-first order kinetics typical of active-site directed chemical modification. Inactivation of ALR2 by [7-C14] phenylglyoxal in the absence of NADPH or NADP+ followed by tryptic digestion resulted in the isolation by HPLC of one major and one minor radioactive peptide. Protein sequencing revealed that the major peptide contained a modified arg268, a residue located in the coenzyme binding site. The minor radioactive peptide and the single radioactive peptide isolated from ALR2 inactivated in the presence of NADP+ contained chemically modified arg293. The arginine residue modified at the active site is positioned to bind the 2'-OH phosphate group of the ribose sugar of the adenine moiety of NADP+. Arg293 is present on the C-terminal loop of ALR2. The enhancement of Arg293 modification by phenylglyoxal in the presence of NADP+ indicates that this C-terminal loop may be involved in the slow conformational change that occurs during the reaction sequence upon coenzyme binding.
authors.affiliation: Department of Biochemistry, Queen's University, Kingston, Ontario, Canada.
authors.forename: T G
authors.initials: TG
authors.lastname: Flynn
authors.forename: T J
authors.initials: TJ
authors.lastname: Kubiseski
mesh_terms: D000449:Aldehyde Reductase / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D001120:Arginine / Q000737:chemistry; D003067:Coenzymes / Q000378:metabolism; D066298:In Vitro Techniques; D007700:Kinetics; D009132:Muscles / Q000201:enzymology; D009249:NADP / Q000378:metabolism; D010658:Phenylglyoxal; D011487:Protein Conformation; D013552:Swine
chemical_list: D003067:Coenzymes; D009249:NADP; D001120:Arginine; D000449:Aldehyde Reductase; D010658:Phenylglyoxal