Kinetic and Spectroscopic Evidence for Active Site Inhibition of Human Aldose Reductase^,

Abstract

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.

Kinetic and Structural Characterization of the Glutathione-binding Site of Aldose Reductase

“…Since, under steady state, this form represents 90 -95% of the enzyme (32), most activesite inhibitors preferentially bind to the E⅐NADP complex. As expected from the complete velocity equation of the enzyme reaction scheme (27,29), such inhibitors do not display competitive inhibition patterns versus the aldehyde, which binds exclusively to the E⅐NADPH binary complex. The binding of uncompetitive inhibitors to the active site of the enzyme has been directly demonstrated by x-ray analysis of E⅐NADP crystals bound to zopolrestat (25) or sorbinil or tolrestat (33).…”

“…In most cases, the nature of inhibition by the glutathione analogs was uncompetitive versus the aldehyde substrate. Although for a simple, one-substrate/one-product reaction scheme, this would suggest that the inhibitors bind to the E⅐S complex, previous experiments have shown that several AR inhibitors such as sorbinil, tolrestat, and zopolrestat that do not display competitive inhibition (26,27) bind exclusively to the active site of the enzyme (27)(28)(29)(30). This is because the rate-limiting step in AR catalysis is the slow isomerization of the E⅐NADP binary complex (31).…”

Characterization of Emodin as a Therapeutic Agent for Diabetic Cataract

“…64 Enzyme solutions were stored at −80 °C and, when thawed, were maintained at 4 °C before use. AKR activity was determined spectrophotometrically by measuring the decrease in absorbance at 340 nm upon oxidation of NADPH.…”

Aldose reductase expression as a risk factor for cataract

“…Expression levels of AKR1B1 in transgenic strains were determined by measuring the rate of DL-glyceraldehyde reduction in the presence of NADPH using lens homogenates in a standard reaction mixture as described previously [29]. Transgene expression levels were also determined by semiquantitative Western blotting as described below.…”