The sorbinil trap: a predicted dead-end complex confirms the mechanism of aldose reductase inhibition.
Kinetic and crystallographic studies have demonstrated that negatively charged aldose reductase inhibitors act primarily by binding to the enzyme complexed with oxidized nicotinamide dinucleotide phosphate (E.NADP(+)) to form a ternary dead-end complex that prevents turnover in the steady state. A recent fluorescence study [Nakano and Petrash (1996) Biochemistry 35, 11196-11202], however, has concluded that inhibition by sorbinil, a classic negatively charged aldose reductase inhibitor, results from binding to the enzyme complexed with reduced cofactor (E.NADPH) and not binding to E.NADP(+). To resolve this controversy, we present transient kinetic data which show unequivocally that sorbinil binds to E.NADP(+) to produce a dead-end complex, the so-called sorbinil trap, which prevents steady-state turnover in the presence of a saturating concentration of aldehyde substrate. The reported fluorescence binding results, which we have confirmed independently, are further shown to be fully consistent with the proposed sorbinil trap mechanism. Our conclusions are supported by KINSIM simulations of both pre-steady-state and steady-state reaction time courses in the presence and absence of sorbinil. Thus, while sorbinil binding indeed occurs to both E.NADPH and E.NADP(+), only the latter dead-end complex shows significant inhibition of the steady-state turnover rate. The effect of tight-binding kinetics on the inhibition patterns observed for zopolrestat, another negatively charged inhibitor, is further examined both experimentally and with KINSIM, with the conclusion that all reported aldose reductase inhibition can be rationalized in terms of binding of an alrestatin-like inhibitor at the active site, with no need to postulate a second inhibitor binding site.
The Harry B. and Aileen Gordon Diabetes Research Laboratory, Molecular Diabetes and Metabolism Section, Department of Pediatrics, Baylor College of Medicine, Houston, Texas 77030, USA. kbohren@bcm.tmc.edu
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D000449:Aldehyde Reductase / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D003198:Computer Simulation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D017022:Flow Injection Analysis; D007093:Imidazoles / Q000494:pharmacology*; D048289:Imidazolidines; D007700:Kinetics; D008956:Models, Chemical; D009249:NADP / Q000378:metabolism*
D004791:Enzyme Inhibitors; D007093:Imidazoles; D048289:Imidazolidines; D009249:NADP; D000449:Aldehyde Reductase; C026411:sorbinil