Carboxymethylation-induced activation of bovine lens aldose reductase.
Carboxymethylation of bovine lens aldose reductase with 10 mM iodoacetate for 1 h at 25 degrees C led to a more than 4-fold increase in kcat. Carboxymethylation led to a 3- to 5-fold increase in Km NADPH and Km D-glyceraldehyde, whereas Km L-glyceraldehyde increased approx. 30-fold. Activation of the enzyme on carboxymethylation was accompanied by a decrease in the sensitivity of the enzyme to inhibition by 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), sorbinil (Kii increased from 0.4 to 109 microM) and NADP (Kis increased from 0.01 to 0.03 mM), but not tolrestat. Activation of the enzyme was almost completely prevented by NADPH and to a lesser extent by DL-glyceraldehyde. Carboxymethylation of the enzyme did not result in the generation of several partially oxidized enzyme species, indicating the absence of partially carboxymethylated forms. Primary deuterium isotope effects on the reduced enzyme were consistent with a preferred ordered kinetic reaction scheme, in which hydride transfer is not rate limiting. The hydride transfer step does not seem to be significantly affected by carboxymethylation, nor do changes in the substrate binding steps seem to contribute to the observed rate enhancement. Increase in the turnover number of the enzyme on carboxymethylation appears to be due to facilitation of the isomerization of the E:NADP binary complex. The differential effect of carboxymethylation on sorbinil and tolrestat suggests distinct inhibitor sites on the enzyme, an S-site that binds sorbinil and a T-site that binds tolrestat.
Liu
S Q
SQ
Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston 77550.
Bhatnagar
A
A
Srivastava
S K
SK
D000449:Aldehyde Reductase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification; D000818:Animals; D002384:Catalysis; D002417:Cattle; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D007093:Imidazoles / Q000494:pharmacology; D048289:Imidazolidines; D007461:Iodoacetates / Q000494:pharmacology; D019807:Iodoacetic Acid; D007700:Kinetics; D007908:Lens, Crystalline / Q000201:enzymology*; D008745:Methylation; D010084:Oxidation-Reduction; D013379:Substrate Specificity
D007093:Imidazoles; D048289:Imidazolidines; D007461:Iodoacetates; D004228:Dithionitrobenzoic Acid; D000449:Aldehyde Reductase; C026411:sorbinil; D019807:Iodoacetic Acid