Kinetic and structural effects of activation of bovine kidney aldose reductase

Abstract

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.

Modulation of aldose reductase activity by aldose hemiacetals

“…The biphasic kinetic behavior occasionally reported for the reduction of different substrates, including D-glucose, by AR and associated with the possible presence of two enzyme forms [16,18,21,25,26], was also observed in the present work, in which all precautions were taken to…”

Mechanism of Human Aldehyde Reductase: Characterization of the Active Site Pocket

“…Release of the alcohol product is followed by an enzyme conformational change, involving in part a nucleotide-clamping loop with release of the oxidized NADP' coenzyme and replacement with NADPH. The nucleotide exchange and its attendant enzyme conformational changes has been shown to be the rate-limiting step for the overall reaction of aldose reductase (Grimshaw et al, 1989;Kubiseski et al, 1992).…”

Specifically Targeted Modification of Human Aldose Reductase by Physiological Disulfides

“…Thus, the so-called "activated" ALR2 generated through apparently different processes such as isomerization (10), glycosylation (11), and thiol-dependent oxidation (12)(13)(14), besides displaying differences in substrate specificity, has a greatly reduced sensitivity to different aldose reductase inhibitors. Indeed, others recently reported the purification of human ALR2 with kinetic properties consistent with those described for an oxidized form of the enzyme (15).…”