Studies on pig aldose reductase. Identification of an essential arginine in the primary and tertiary structure of the enzyme.
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.
Kubiseski
T J
TJ
Department of Biochemistry, Queen's University, Kingston, Ontario, Canada.
Green
N C
NC
Borhani
D W
DW
Flynn
T G
TG
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
D010446:Peptide Fragments; D012997:Solvents; D009249:NADP; D001120:Arginine; D000449:Aldehyde Reductase; D010658:Phenylglyoxal