Kinetic and spectroscopic evidence for active site inhibition of human aldose reductase.
Aldose reductase is an NADPH-dependent oxidoreductase that catalyzes the reduction of a variety of aldehydes and carbonyls, including monosaccharides. Intense interest in the discovery and characterization of inhibitors has developed since the action of this enzyme has been linked to the pathogenesis of some diabetic complications. Since past studies indicated that most inhibitors act noncompetitively or uncompetitively versus substrate in the direction of aldehyde reduction, it was assumed that they bind at one or more sites distinct from the active site. However, the crystal structure of aldose reductase complexed with inhibitor [Wilson et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 9847-9851] unambiguously revealed the inhibitor bound at the active site. The present study was undertaken to address this apparent discrepancy. Using a fluorometric assay, it was determined that zopolrestat, an acetic acid-type inhibitor, bound to aldose reductase complexed with either NADPH or NADP+. In contrast, the spirohydantoin-type inhibitor sorbinil demonstrated preferential binding to the binary enzyme.NADPH complex. Prior incubation of the enzyme.NADPH complex with zopolrestat prevented subsequent sorbinil binding. These results, together with the published structure of the ternary enzyme.NADPH.zopolrestat complex, are consistent with the conclusion that both sorbinil and zopolrestat bind at the active site. We propose that mixed inhibition patterns previously observed with sorbinil are due to inhibitor binding to both structural isomers of the enzyme.NADPH complex. Similar patterns for inhibition by zopolrestat are due to tight binding of the inhibitor. Substrate inhibition in the direction of aldehyde reduction occurs as a result of substrate binding to the enzyme.NADP+ complex.
Nakano
T
T
Department of Ophthalmology and Visual Sciences, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Petrash
J M
JM
D000449:Aldehyde Reductase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D052160:Benzothiazoles; D001665:Binding Sites / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000378:metabolism / Q000494:pharmacology*; D005985:Glyceraldehyde / Q000378:metabolism; D006801:Humans; D006827:Hydantoins / Q000494:pharmacology; D007093:Imidazoles / Q000494:pharmacology*; D048289:Imidazolidines; D007700:Kinetics; D010793:Phthalazines / Q000494:pharmacology*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D013844:Thiazoles / Q000494:pharmacology*
D052160:Benzothiazoles; D004791:Enzyme Inhibitors; D006827:Hydantoins; D007093:Imidazoles; D048289:Imidazolidines; D010793:Phthalazines; D011994:Recombinant Proteins; D013844:Thiazoles; C067172:zopolrestat; D005985:Glyceraldehyde; D000449:Aldehyde Reductase; C026411:sorbinil