| Popis: |
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. |
