Residues affecting the catalysis and inhibition of rat lens aldose reductase.
Aldose reductase (AR), the first enzyme of the polyol pathway, has been implicated in diabetic complications. Results of recent clinical studies have shown that compounds that inhibit aldose reductase (ARIs) and block the flux of glucose through the polyol pathway have provided benefit to diabetic neuropathic patients. Since many ARIs show broad substrate specificity, emphasis on the structure-function properties of the AR enzyme will help in the refinement and design of future inhibitors. To this end, catalysis and inhibition of rat lens aldose reductase was examined following site-directed mutagenesis. Replacement of tyrosine 48 with phenylalanine (Y48F) resulted in an enzyme form with less than 0.25% activity with DL-glyceraldehyde and no detectable activity with p-nitrobenzaldehyde or xylose, although circular dichroism spectra and NADPH binding affinity were similar to wild-type AR. Mutation of histidine 110 to glutamine (H110Q) also resulted in a less active protein with an approximate 3-fold decrease in kcat for the reduction of DL-glyceraldehyde; slight or no activity was measured with other substrates and an increase of 195-fold over wild type was observed in the Km for glyceraldehyde. H110Q was less sensitive to inhibition by aldose reductase inhibitors. The most dramatic change was seen with imeristat, which showed an 1800-fold increase in IC50. Mutation of cysteine 298 to serine (C298S) affected enzyme function by increasing kcat 2- to 4-fold and increasing Km 15- to 48-fold, with DL-glyceraldehyde, p-nitrobenzaldehyde or xylose as substrates. As a result kcat/Km, catalytic efficiency, dropped to approx. 10% of control. Inhibition of C298S was not noticeably different from wild type. Substitution of histidine 187 or 200 with glutamine (H187Q, H200Q) had little effect on AR catalysis or inhibition. Based on structural and mutagenesis studies of human AR and the conservation of amino acids between human and rat, these data would indicate that Y48, H110, and C298 are important residues in the active site of rat AR and that Y48 is most likely the proton donor during substrate reduction by rat lens aldose reductase. In addition, these studies indicate that mutagenesis of H110 also affects aldose reductase inhibition.
Carper
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DA
National Eye Institute, National Institutes of Health, Bethesda, MD 20892.
Hohman
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TC
Old
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SE
D000449:Aldehyde Reductase / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D002384:Catalysis; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D005973:Glutamine; D006639:Histidine; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007908:Lens, Crystalline / Q000201:enzymology*; D016297:Mutagenesis, Site-Directed; D009249:NADP / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D011994:Recombinant Proteins; D005973:Glutamine; D006639:Histidine; D009249:NADP; D000449:Aldehyde Reductase