ANION-BINDING SITE IN HUMAN ALDOSE REDUCTASE - MECHANISTIC IMPLICATIONS FOR THE BINDING OF CITRATE, CACODYLATE, AND GLUCOSE-6-PHOSPHATE

Aldose reductase is a NADPH-dependent aldo-keto reductase involved in the pathogenesis of some diabetic and galactosemic complications. The published crystal structure of human aldose reductase [Wilson et al. ( 1992) Science 257, 81-84] contains a hitherto unexplained electron den sity positioned within the active site pocket facing the nicotinamide ring of the NADPH and other key active site residues (Tyr48, His110, a nd Cys298). In this paper we identify the electron density as citrate, which is present in the crystallization buffer (pH 5.0), and provide confirmatory evidence by both kinetic and crystallographic experiments . Citrate is an uncompetitive inhibitor in the forward reaction with r espect to aldehyde (reduction of aldehyde), while it is a competitive inhibitor with respect to alcohol in the backward reaction (oxidation of alcohol), indicating that it interacts with the enzyme-NADP(+)-prod uct complex. Citrate can be replaced in the crystalline enzyme complex by cacodylate or glucose 6-phosphate; the structure of each of these complexes shows the specific molecule bound in the active site. All of the structures have been determined to a nominal resolution of 1.76 A ngstrom and refined to R-factors below 18%. While cacodylate can be bo und within the active site under the crystallization conditions, it do es not inhibit the wild-type enzyme in solution. Glucose 6-phosphate, however, is a substrate for aldose reductase. The similar location of the negative charges of citrate, cacodylate, and glucose 6-phosphate w ithin the active site suggests an anion-binding site delineated by the C4N of nicotinamide, the OH of Tyr48, and the NE Of His110. The locat ion of citrate binding in the active site leads to a plausible catalyt ic mechanism for aldose reductase.