Abstract
In order to study the potential role of cysteinyl residues in catalysis and inhibition of human aldose reductase, mutants containing cysteine to serine substitution at positions 80 (ALR2:C80S), 298 (ALR2:C298S), and 303 (ALR2:C303S) were constructed. Mutation of Cys298 resulted in the most profound changes, as ALR2:C298S displayed 4- to 5-fold elevation in K'm(NADPH), K'm(DL-glyceraldehyde), and kcat(DL-glyceraldehyde) relative to wild type aldose reductase as well as a 10-fold higher Ki for the aldose reductase inhibitor sorbinil. Wild type and mutant reductases were equally sensitive to tolrestat, a structurally different reductase inhibitor. Carboxymethylation of the wild type enzyme or the C80S and C303S mutants led to a modest decrease in kcat as well as an increase in K'm(DL-glyceraldehyde) and Ki(sorbinil). These parameters were not significantly changed when ALR2:C298S was subjected to carboxymethylation. Lithium sulfate caused activation of ALR2:WT, C80S, and C303S but did not significantly affect the activity of ALR2:C298S. The differential sensitivity of wild type and mutant reductases to inhibition by sorbinil and tolrestat, before and after carboxymethylation, indicates that these inhibitors bind at different sites. These results suggest that Cys-298 is present near the active site and constitutes a regulatory group which controls the catalytic activity and inhibitor sensitivity of the enzyme.
Highlights
In order to study the potential rolecyosfteinyl resi- and other aldo-sugars to their corresponding sugar alcohols dues incatalysisand inhibition of human aldose reduocr- polyols [1,2]
ALR2:WT, CSOS, andC303S but did notsignificantly specific aldose reductase inhibitors will depend on an underaffect the activity of ALR2:C298S
These results suggest that Cys-298 is present near the active site and constitutes a regustanding of the mechanism of catalysis of the enzyme and synthesis of mechanism-based inhibitors or transition state analogs
Summary
Amino Acid Sequence Analysis of Recombinant Aldose RedwtaseAliquots of recombinant aldose reductase taken from selected chromatofocusing fractions (greater than 90% pure) were subjected to aldose reductase and cross-reacted with antibodies to bovine lens aldose reductase (Fig. 1, lanes 2-6) The abundance of this polypeptide increased significantly in cells cultured for 2 h followingthe addition of 1mM IPTG (compare Fig. 1,lanes automated Edman degradation using an Applied Biosystems, Inc. 2 and 3). The enzyme solution was maintained in 10mM imidazole-HC1,pH 7.0, containing 5 mM @-mercaptoethanol.Homogeneity of the purified enzyme was established by the movement of a single protein band on reducing tions from cultures containingpMON5997 revealed that most of the aldose reductase expressed in E. coli can be released by osmotic shock. Data were considered statistically significant when the p value was
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