Mechanism of Aldose Reductase Inhibition: Binding of NADP+/NADPH and Alrestatin-like Inhibitors

Torsten Ehrig, Franklyn G. Prendergast, Kurt M. Bohren, Kenneth H. Gabbay, Kenneth H. Gabbay

Research output: Contribution to journalArticle

82 Scopus citations

Abstract

Aldose reductase enfolds NADP+/NADPH via a complex loop mechanism, with cofactor exchange being the rate-limiting step for the overall reaction. This study measures the binding constants of these cofactors in the wild-type enzyme, as well as a variety of active-site mutants (C298A, Y48H, Y48F, Y209F, H110A, W219A, and W20A), and seeks to identify the binding site and mechanism of the aldose reductase inhibitor alrestatin in the recombinant human enzyme. All the mutant enzymes, regardless of their enzyme activities, have normal or only slightly elevated coenzyme binding constants, suggesting a tertiary structure similar to that of the wild-type enzyme. Binding of alrestatin was detected by fluorescence assays, and by an ultrafiltration assay which measures the fraction of unbound alrestatin. Alrestatin binds preferentially to the enzyme/NADP+ complex, consistent with the steady-state inhibition pattern. Alrestatin binding and enzyme inhibition were abolished in the Tyr48 mutants Y48F and Y48H, implicating the positively charged anion well formed by the Asp43/Lys77+/Tyr480/NADP+ complex in inhibitor binding (Harrison et al., 1994; Bohren et al., 1994). The enzyme mutant W20A severely affected the inhibitory potencies of a variety of commercially developed aldose reductase inhibitors (zopolrestat, tolrestat, FK366, AL1576, alrestatin, ponalrestat, and sorbinil). Inhibition by citrate, previously shown to bind to the positively charged anion well, was not affected by this mutation. Inhibitors with flexible double aromatic ring systems (zopolrestat, FK366, and ponalrestat) were less affected than others possessing a single aromatic ring system, suggesting that the additional pharmacophor ring system stabilizes the inhibitor by interaction at some other hydrophobic site. These findings 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 avidity of binding.

Original languageEnglish (US)
Pages (from-to)7157-7165
Number of pages9
JournalBiochemistry
Volume33
Issue number23
DOIs
StatePublished - Jun 1 1994

ASJC Scopus subject areas

  • Biochemistry

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