title: Studies on pig aldose reductase. Identification of an essential arginine in the primary and tertiary structure of the enzyme.
abstract: Reaction of pig muscle aldose reductase with phenylglyoxal resulted in the chemical modification of 2 arginine residues with accompanying loss of catalytic activity. The amino acid sequences of radioactive peptides resulting from the reaction of aldose reductase with [14C]phenylglyoxal followed by tryptic digestion and high performance liquid chromatography separation allowed identification of the modified arginine residues as R268 and R293. In the presence of the coenzyme NADP+, R268 is protected from modification by phenylglyoxal, while R293 becomes hyper-reactive. Phenylglyoxal modification of aldose reductase is slowed 3-fold by the presence of the coenzyme analog ADPRP; however, both arginines are still modified. These chemical modification results are in complete accord with the previously determined crystal structures of human and porcine aldose reductase complexed with NADPH, NADP+, and ADPRP. These structures indicate that R268 is located at the adenosine binding site, salt bridged to the 2'-phosphate group of NADP(H) and ADPRP. Arginine 293 is near the surface of the enzyme and is part of the C-terminal loop. In the apoenzyme or the ADPRP complex, R293 is partially protected by loop 7; upon binding NADP(H), loop 7 folds down over the coenzyme, thus exposing R293 to solvent. Our modification studies provide further evidence of the conformational change that occurs during the aldose reductase catalytic cycle.
authors.affiliation: Department of Biochemistry, Queen's University, Kingston, Ontario, Canada.
authors.forename: T J
authors.initials: TJ
authors.lastname: Kubiseski
authors.forename: N C
authors.initials: NC
authors.lastname: Green
authors.forename: D W
authors.initials: DW
authors.lastname: Borhani
authors.forename: T G
authors.initials: TG
authors.lastname: Flynn
mesh_terms: D000449:Aldehyde Reductase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine; D001665:Binding Sites; D007700:Kinetics; D008433:Mathematics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009132:Muscles / Q000201:enzymology; D009249:NADP / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010658:Phenylglyoxal / Q000378:metabolism / Q000494:pharmacology; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D012997:Solvents; D013552:Swine
chemical_list: D010446:Peptide Fragments; D012997:Solvents; D009249:NADP; D001120:Arginine; D000449:Aldehyde Reductase; D010658:Phenylglyoxal