Bovine kidney aldose reductase (ALR2) displays substrate inhibition by aldehyde substrates that is uncompetitive versus NADPH when allowance is made for nonenzymic reaction of the aldehyde with the adenine moiety of NADPH. A time-dependent increase in substrate inhibition observed in product versus time plots for reduction of short-chain aldoses containing an enolizable alpha-proton, but not for p-nitrobenzaldehyde, is shown to be consistent with a model in which rapidly reversible inhibition due to formation of the dead-end E-NADP-glycolaldehyde complex is combined with the formation at the enzyme active site of a tightly-bound covalent NADP-glycolaldehyde adduct. Quantitative analysis of reaction time courses for ALR2-catalyzed reduction of glycolaldehyde using NADPH or the 3-acetylpyridine analogue, (AP)ADPH, yields values of the forward and reverse rate constants for ALR2-mediated adduct formation that agree with the values determined in the absence of glycolaldehyde turnover. Substrate inhibition is only partial, indicating that reaction can occur via an alternate pathway at high [glycolaldehyde]. Kinetic evidence for a slow isomerization of the E-NADP complex at pH 8.0 is used to explain the similar V/Et values observed for glycolaldehyde reduction at pH 7.0 using NADPH, (AP)ADPH, and the hypoxanthine analogue N(Hx)DPH. The practical implications of these results for kinetics studies of aldose reductase are discussed.

Aldose reductase, purified to homogeneity from bovine kidney, is converted in a temperature-dependent process from a low-Km/low-Vmax form to a high-Km/high-Vmax form of the enzyme. Activation, which results in significant changes in the protein secondary structure, as detected by fluorescence spectroscopy, circular dichroism, and thiol modification with 5,5'-dithiobis(2-nitrobenzoic acid), has no effect on the apparent Mr, pI, or homogeneity of the enzyme, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and agarose isoelectric focusing. Vmax, which varied less than 3-fold for a series of aldehyde substrates with either activation state of the enzyme, increased an average of (17 +/- 4)-fold upon activation of the enzyme. V/Kaldehyde increased or decreased up to 4-fold, depending on the substrate. Activation desensitized the enzyme to inhibition by aldose reductase inhibitors, with the apparent Ki value increasing from 2-fold for Epalrestat [ONO-2235, (E)-3-(carboxymethyl)-(E)-5-[2-methyl-3-phenylpropenylidene]-rhoda nine] to 200-fold for AL-1576 (spiro [2,7-difluorofluorene-9,4'-imidazolidine]-2',5'-dione). Biphasic double-reciprocal plots for the aldehyde substrates and biphasic Dixon plots for inhibition by AL-1576 and Statil [ICI-128,436; 3-[(4-bromo-2-fluorobenzyl)-4-oxo-3H-phthalazin-l-ylacetic acid], observed during the course of activation, are quantitatively accounted for by the individual contributions of the two enzyme forms. On the basis of an analysis of the kinetic data, a mechanism is proposed in which isomerization of the free enzyme limits the rate of the forward reaction for the unactivated enzyme and is the primary step affected by activation.

Reaction of glycolaldehyde with the binary E-NADP complex of bovine kidney aldose reductase (ALR2) produces an enzyme-bound chromophore whose absorbance (lambd max 341 nm) and fluorescence (lambda ex max 341 nm; lambda emit max 421 nm) properties are distinct from those of NADPH or E.NADPH yet are consistent with the proposed covalent adduct structure [1,4-dihydro-4-(1-hydroxy-2-oxoethyl)nicotinamide adenine dinucleotide phosphate]. The kinetics of adduct formation, both in solution and at the enzyme active site, support a mechanism involving rate-determining enolization of glycolaldehyde at high [NADP+] or [E.NADP]. At low [NADP+] or [E.NADP] the reaction is second-order overall, but the ALR2-mediated reaction displays saturation by glycolaldehyde due to competition of the aldehyde (plus hydrate) and enol for E.NADP. Measurement of the pre-steady-state burst of E-adduct formation confirms that glycolaldehyde enol is the reactive species and gives a value of 1.3 x 10(-6) for Kenol = [enol]/[( aldehyde] + [hydrate]), similar to that determined by trapping the enol with I3-. At the ALR2 active site, the rate of adduct formation is enhanced 79,000-fold and the adduct is stabilized greater than or equal to 13,000-fold relative to the reaction with NADP+ in solution. A portion of this enhancement is ascribed to specific interaction of NADP+ with the enzyme since the 3-acetylpyridine analogue, (AP)ADP+, gives values that are 15-200-fold lower. Additional evidence for strong interaction of ALR2 with both NADP+ and NADPH is reported. Yet, because dissociation of adduct is slow, catalysis of the overall adduct formation reaction by ALR2 is less than or equal to 67-fold.

No abstract