MECHANISM OF ALDOSE REDUCTASE INHIBITION - BINDING OF NADP+ NADPH ANDALRESTATIN-LIKE INHIBITORS/

Aldose reductase enfolds NADP(+)/NADPH via a complex loop mechanism, w ith cofactor exchange being the rate-limiting step for the overall rea ction. This study measures the binding constants of these cofactors in the wild-type enzyme, as well as a variety of active-site mutants (C2 98A, Y48H, Y48F, Y209F, H110A, W219A, and W20A), and seeks to identify the binding site and mechanism of the aldose reductase inhibitor alre statin in the recombinant human enzyme. All the mutant enzymes, regard less of their enzyme activities, have normal or only slightly elevated coenzyme binding constants, suggesting a tertiary structure similar t o that of the wild-type enzyme. Binding of alrestatin was detected by fluorescence assays, and by an ultrafiltration assay which measures th e fraction of unbound alrestatin. Alrestatin binds preferentially to t he enzyme/NADP(+) complex, consistent with the steady-state inhibition pattern. Alrestatin binding and enzyme inhibition were abolished in t he Tyr48 mutants Y48F and Y48H, implicating the positively charged ani on well formed by the Asp43(-)/Lys77(+)/Tyr48(0)/NADP(+) complex in in hibitor binding (Harrison et al., 1994; Bohren et al., 1994). The enzy me mutant W20A severely affected the inhibitory potencies of a variety of commercially developed aldose reductase inhibitors (zopolrestat, t olrestat, FK366, AL 1576, alrestatin, ponalrestat, and sorbinil). Inhi bition by citrate, previously shown to bind to the positively charged anion well, was not affected by this mutation. Inhibitors with flexibl e double aromatic ring systems (zopolrestat, FK366, and ponalrestat) w ere less affected than others possessing a single aromatic ring system , suggesting that the additional pharmacophor ring system stabilizes t he inhibitor by interaction at some other hydrophobic site. These find ings indicate that the inhibitors require a negative charge to anchor to the anion well, and stacking of their aromatic ring systems against the Trp20 residue in the active site to stabilize and increase the av idity of binding.