1,2-Benzothiazine 1,1-dioxide carboxylate derivatives as novel potent inhibitors of aldose reductase

Due to the importance of aldose reductase (ALR2) as a potential drug target in the treatment of diabetic complications, there are increasing interests in design and synthesis of ALR2 inhibitors. Here, we prepared 1,2-benzothiazine 1,1-dioxide acetic acid derivatives and investigated their inhibition activity. Most of these derivatives were found to be active with IC(50) values ranging from 0.11 μM to 10.42 μM, and compound 8d, 2-[2-(4-bromo-2-fluorobenzyl)-1,1-dioxido-2H-1,2-benzothiazin-4(3H)-ylidene]acetic acid, showed the most potent inhibition activity. Further, SAR and docking studies suggest that in comparison with the α,β-unsaturated derivatives, the saturated carboxylic acid derivatives had a greater binding affinity with the enzyme and thus an enhanced inhibition activity. Therefore, development of more powerful ARIs based on benzothiazine 1,1-dioxide by stereo-controlled synthesis could be expected.     

The effects of zenarestat, an aldose reductase inhibitor, on minimal F-wave latency and nerve blood flow in streptozotocin-induced diabetic rats

The effects of zenarestat, 3-(4-bromo-2-fluorobenzyl)-7-chloro-3,4-dihydro-2,4-dioxo-1(2H)-quinazolineacetic acid, an aldose reductase inhibitor (ARI), on F-wave conduction abnormalities, nerve blood flow (NBF) reduction and sorbitol accumulation were studied in streptozotocin-induced diabetic rats. Two weeks after the induction of diabetes, zenarestat was given once a day for two weeks. In diabetic control rats, marked accumulation of sorbitol, reduction of NBF and prolongation of minimal F-wave latency (FWL) were observed as compared to normal rats. Zenarestat, at a dose of 32 mg/kg, inhibited sorbitol concentration to nearly the normal rat level and significantly improved not only NBF but also minimal FWL. At a dose of 3.2 mg/kg, sorbitol accumulation was inhibited by approximately 40% and there was a tendency to increase in NBF; however, minimal FWL was not improved at all. These data suggest that a highly inhibition of the nerve sorbitol accumulation is requisite for the treatment of diabetic peripheral neuropathy.     

Kinetic and structural effects of activation of bovine kidney aldose reductase

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.     

Catalytic effectiveness of human aldose reductase. Critical role of C-terminal domain.

Human aldose reductase and aldehyde reductase are members of the aldo-keto reductase superfamily that share three domains of homology and a nonhomologous COOH-terminal region. The two enzymes catalyze the NADPH-dependent reduction of a wide variety of carbonyl compounds. To probe the function of the domains and investigate the basis for substrate specificity, we interchanged cDNA fragments encoding the NH2-terminal domains of aldose and aldehyde reductase. A chimeric enzyme (CH1, 317 residues) was constructed in which the first 71 residues of aldose reductase were replaced with first 73 residues of aldehyde reductase. Catalytic effectiveness (kcat/Km) of CH1 for the reduction of various substrates remained virtually identical to wild-type aldose reductase, changing a maximal 4-fold. Deletion of the 13-residue COOH-terminal end of aldose reductase, yielded a mutant enzyme (AR delta 303-315) with markedly decreased catalytic effectiveness for uncharged substrates ranging from 80- to more than 600-fold (average 300-fold). The KmNADPH of CH1 and AR delta 303-315 were nearly identical to that of the wild-type enzyme indicating that cofactor binding is unaffected. The truncated AR delta 303-315 displayed a NADPH/D isotope effect in kcat and an increased D(kcat/Km) value for DL-glyceraldehyde, suggesting that hydride transfer has become partially rate-limiting for the overall reaction. We conclude that the COOH-terminal domain of aldose reductase is crucial to the proper orientation of substrates in the active site.     

Kinetic and structural characterization of the glutathione-binding site of aldose reductase

Aldose reductase (AR), a member of the aldo-keto reductase superfamily, has been implicated in the etiology of secondary diabetic complications. However, the physiological functions of AR under euglycemic conditions remain unclear. We have recently demonstrated that, in intact heart, AR catalyzes the reduction of the glutathione conjugate of the lipid peroxidation product 4-hydroxy-trans-2-nonenal (Srivastava, S., Chandra, A., Wang, L., Seifert, W. E., Jr., DaGue, B. B., Ansari, N. H., Srivastava, S. K., and Bhatnagar, A. (1998) J. Biol. Chem. 273, 10893-10900), consistent with a possible role of AR in the metabolism of glutathione conjugates of aldehydes. Herein, we present several lines of evidence suggesting that the active site of AR forms a specific glutathione-binding domain. The catalytic efficiency of AR in the reduction of the glutathione conjugates of acrolein, trans-2-hexenal, trans-2-nonenal, and trans,trans-2,4-decadienal was 4-1000-fold higher than for the corresponding free alkanal. Alterations in the structure of glutathione diminished the catalytic efficiency in the reduction of the acrolein adduct, consistent with the presence of specific interactions between the amino acid residues of glutathione and the AR active site. In addition, non-aldehydic conjugates of glutathione or glutathione analogs displayed active-site inhibition. Molecular dynamics calculations suggest that the conjugate adopts a specific low energy configuration at the active site, indicating selective binding. These observations support an important role of AR in the metabolism of glutathione conjugates of endogenous and xenobiotic aldehydes and demonstrate, for the first time, efficient binding of glutathione conjugates to an aldo-keto reductase.     

Characterization of the glutathione binding site of aldose reductase

Despite extensive investigations, the physiological role of the polyol pathway enzyme–aldose reductase (AR) remains obscure. While the enzyme reduces glucose in vivo and in vitro, kinetic and structural studies indicate inefficient carbohydrate binding to the active site of the enzyme. The active site is lined by hydrophobic residues and appears more compatible with the binding of medium- to long-chain aliphatic aldehydes or hydrophobic aromatic aldehydes. In addition, our recent studies show that glutathione (GS) conjugates are also reduced efficiently by the enzyme. For instance, the GS conjugate of acrolein is reduced with a catalytic efficiency 1000-fold higher than the parent aldehyde, indicating specific recognition of glutathione by the active site residues of AR. An increase in the catalytic efficiency upon glutathiolation was also observed with trans-2-nonenal, trans-2-hexenal and trans, trans-2,4-decadienal, establishing that enhancement of catalytic efficiency was specifically due to the glutathione backbone and not specific to the aldehyde. Structure–activity relationships with substitution or deletion of amino acids of GSH indicated specific interactions of the active site with γ-Glu1 and Cys of GSH. Molecular modeling revealed that the glutathione–propanal conjugate could bind in two distinct orientations. In orientation 1, γ-Glu1 of the conjugate interacts with Trp20, Lys21 and Val47, and Gly3 interacts with Ser302 and Leu301, whereas in orientation 2, the molecule is inverted with γ-Glu1 interacting with Ser302, and Leu301. Taken together, these data suggest that glutathiolation of aldehydes enhances their compatibility with the AR active site, which may be of physiological significance in detoxification of endogenous and xenobiotic aldehydes.     

Characterization of the glutathione binding site of aldose reductase

Despite extensive investigations, the physiological role of the polyol pathway enzyme-aldose reductase (AR) remains obscure. While the enzyme reduces glucose in vivo and in vitro, kinetic and structural studies indicate inefficient carbohydrate binding to the active site of the enzyme. The active site is lined by hydrophobic residues and appears more compatible with the binding of medium- to long-chain aliphatic aldehydes or hydrophobic aromatic aldehydes. In addition, our recent studies show that glutathione (GS) conjugates are also reduced efficiently by the enzyme. For instance, the GS conjugate of acrolein is reduced with a catalytic efficiency 1000-fold higher than the parent aldehyde, indicating specific recognition of glutathione by the active site residues of AR. An increase in the catalytic efficiency upon glutathiolation was also observed with trans-2-nonenal, trans-2-hexenal and trans, trans-2,4-decadienal, establishing that enhancement of catalytic efficiency was specifically due to the glutathione backbone and not specific to the aldehyde. Structure-activity relationships with substitution or deletion of amino acids of GSH indicated specific interactions of the active site with gamma-Glu1 and Cys of GSH. Molecular modeling revealed that the glutathione-propanal conjugate could bind in two distinct orientations. In orientation 1, gamma-Glu1 of the conjugate interacts with Trp20, Lys21 and Val47, and Gly3 interacts with Ser302 and Leu301, whereas in orientation 2, the molecule is inverted with gamma-Glu1 interacting with Ser302, and Leu301. Taken together, these data suggest that glutathiolation of aldehydes enhances their compatibility with the AR active site, which may be of physiological significance in detoxification of endogenous and xenobiotic aldehydes.     

Structure of a glutathione conjugate bound to the active site of aldose reductase

Aldose reductase (AR) is a monomeric NADPH-dependent oxidoreductase that catalyzes the reduction of aldehydes, ketones, and aldo-sugars. AR has been linked to the development of hyperglycemic injury and is a clinical target for the treatment of secondary diabetic complications. In addition to reducing glucose, AR is key regulator of cell signaling through it's reduction of aldehydes derived from lipoproteins and membrane phospholipids. AR catalyzes the reduction of glutathione conjugates of unsaturated aldehydes with higher catalytic efficiency than free aldehydes. The X-ray structure of human AR holoenzyme in complex with the glutathione analogue S-(1,2-dicarboxyethyl) glutathione (DCEG) was determined at a resolution of 1.94 A. The distal carboxylate group of DCEG's dicarboxyethyl moiety interacted with the conserved AR anion binding site residues Tyr48, His110, and Trp111. The bound DCEG's glutathione backbone adopted the low-energy Y-shape form. The C-terminal carboxylate of DCEG glutathione's glycine formed hydrogen bonds to Leu301 and Ser302, while the remaining interactions between DCEG and AR were hydrophobic, permitting significant flexibility of the AR and glutathione (GS) analogue interaction. The observed conformation and interactions of DCEG with AR were consistent with our previously published molecular dynamics model of glutathionyl-propanal binding to AR. The current structure identifies major interactions of glutathione conjugates with the AR active-site residues.     

Kinetics and molecular docking studies of kaempferol and its prenylated derivatives as aldose reductase inhibitors

Aldose reductase inhibitors (ARIs) suppressing the hyperglycemia-induced polyol pathway have been provided as potential therapeutic candidates in the treatment and prevention of diabetic complications. Based upon structure-activity relationships of desmethylanhydroicaritin (1) and sophoflavescenol (2) as promising ARIs, 3,4'-dihydroxy flavonols with a prenyl or lavandulyl group at the C-8 position and a hydroxyl or methoxy group at the C-5 position are important for aldose reductase (AR) inhibition. In order to prove the above results, a combination of computational prediction and enzyme kinetics has begun to emerge as an effective screening technique for the potential. In the present study, we predicted the 3D structure of AR in rat and human using a docking algorithm to simulate binding between AR and prenylated flavonoids (1 and 2) and kaempferol (3) and scrutinized the reversible inhibition of AR by these ARIs. Docking simulation results of 1-3 demonstrated negative binding energies (Autodock 4.0=-9.11 to -7.64 kcal/mol; Fred 2.0=-79.54 to -51.84 kcal/mol) and an additional hydrogen bond through Phe122 and Trp219, in addition to the previously proposed interaction of AR and phenolics through Trp20, Tyr48, His110, and Trp111 residues, indicating that the presence of 8-prenyl and 5-methyl groups might potentiate tighter binding to the active site of the enzyme and more effective AR inhibitors. Moreover, types of AR inhibition were different depending on the presence or absence of the 8-prenyl group, in that 1 and 2 are mixed inhibitors with respective Ki values of 0.69 μM and 0.94 μM, while 3 showed noncompetitive inhibition with a Ki value of 4.65 μM. The present study suggests that an effective strategy for screening potential ARIs could be established by predicting 3D structural conformation of prenylated flavonoids and the orientation within the enzyme as well as by simultaneously determining the mode of enzyme inhibition.     

Comparison of the catalytic and inhibitory properties of Pachysolen tannophilus xylose reductase to rat lens aldose reductase

Summary The catalytic and inhibitory profiles of xylose reductase isolated from the yeast Pachysolen tannophilus (PTXR) are compared to those of aldose reductase (AR) obtained form rat lens. While both PTXR and rat lens AR are NADPH-specific enzymes and have an affinity for a variety of substrates such as d-xylose, d,l-glyceraldehyde, and 4-nitrobenzaldehyde, the enzymes differ in their substrate affinity profiles. Also, PTXR is not inhibited by standard inhibitors of AR thus supporting a hypothesis that this enzyme may not possess the inhibitor binding site found in rat lens AR. Offprint requests to: J. DeRuiter     

Mechanistic basis for nonlinear kinetics of aldehyde reduction catalyzed by aldose reductase

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.     

Virtual screening for inhibitors of human aldose reductase

The inhibition of aldose reductase (AR) provides an interesting strategy to prevent the complications of chronic diabetes. Although a large number of different AR inhibitors are known, very few of these compounds exhibit sufficient efficacy in clinical trials. We performed a virtual screening based on the ultrahigh resolution crystal structure of the inhibitor IDD594 in complex with human AR. AR operates on a large scale of structurally different substrates. To achieve this pronounced promiscuity, the enzyme can adapt rather flexibly to its substrates. Likewise, it has a similar adaptability for the binding of inhibitors. We applied a protocol of consecutive hierarchical filters to search the Available Chemicals Directory. In the first selection step, putative ligands were chosen that exhibit functional groups to anchor the anion-binding pocket of AR. Subsequently, a pharmacophore model based on the binding geometry of IDD594 and the mapping of the binding pocket in terms of putative "hot spots" of binding was applied as a second consecutive filter. In a third and final filtering step, the remaining candidate molecules were flexibly docked into the binding pocket of IDD594 with FlexX and ranked according to their estimated DrugScore values. Out of 206 compounds selected by this search and complemented by a cluster analysis and visual inspection, 9 compounds were selected and subjected to biological testing. Of these, 6 compounds showed IC50 values in the micromolar range. According to the proposed binding mode, the two inhibitors BTB02809 (IC50 = 2.4 +/- 0.5 microM) and JFD00882 (IC50 = 4.1 +/- 1.0 microM) both place a nitro group into the hydrophobic specificity pocket of human AR in an orientation coinciding with the position of the bromine atom of IDD594. The interaction of this Br with Thr113 has been identified as a key feature that is responsible for selectivity enhancement.     

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.     

Methotrexate, a high-affinity pseudosubstrate of dihydrofolate reductase.

Investigations have been made of the slow, tight-binding inhibition by methotrexate of the reaction catalyzed by dihydrofolate reductase from Streptococcus faecium A. Quantitative analysis has shown that progress curve data are in accord with a mechanism that involves the rapid formation of an enzyme-NADPH-methotrexate complex that subsequently undergoes a relatively slow, reversible isomerization reaction. From the Ki value for the dissociation of methotrexate from the E-NADPH-methotrexate complex (23 nM) and values of 5.1 and 0.013 min-1 for the forward and reverse rate constants of the isomerization reaction, the overall inhibition constant for methotrexate was calculated to be 58 pM. The formation of an enzyme-methotrexate complex was demonstrated by means of fluorescence quenching, and a value of 0.36 muM was determined for its dissociation constant. The same technique was used to determine dissociation constants for the reaction of methotrexate with the E-NADP and E-NADPH complexes. The results indicate that in the presence of either NADPH or NADP there is enhancement of the binding of methotrexate to the enzyme. It is proposed that methotrexate behaves as a pseudosubstrate for dihydrofolate reductase.     

Inhibitory effect of ursolic acid derivatives on recombinant human aldose reductase

Aldose reductase (AR) is the first enzyme in the polyol pathway. AR has been reported to play an important role in the pathogenesis of diabetic complications. Ursolic acid and fourteen synthetic derivatives with ursane skeleton were tested for recombinant human aldose reductase (rhAR) inhibitory activity for development of diabetic complications. Among them, N-(3β-hydroxyurs-12-en-28-oyl)-4-aminobutyric acid (XV) showed most potent rhAR inhibitory activity in vitro. Inhibition mode of N-(3β-hydroxyurs-12-en-28-oyl)-4-aminobutyric acid (XV) was tested uncompetitively by kinetic analysis using the Lineweaver-Burk plots. Ursolic acid derivative N-(3β-hydroxyurs-12-en-28-oyl)-4-aminobutyric acid is able to inhibit rhAR uncompetitively and could be offered as a lead compound for AR inhibition.     

In vitro aldose reductase inhibitory activity of some flavonyl-2,4-thiazolidinediones

Aldose reductase (AR) is implicated to play a critical role in diabetes and cardiovascular complications because of the reaction it catalyzes. AR enzyme appears to be the key factor in the reduction of glucose to sorbitol. Synthesis and accumulation of sorbitol in cells due to AR activity is the main cause of diabetic complications, such as diabetic cataract, retinopathy, neuropathy and nephropathy. Aldose reductase inhibitors have been found to prevent sorbitol accumulation in tissues. Numerous compounds have been prepared in order to improve the pharmacological prophile of inhibition of aldose reductase enzyme. In this study, seventeen flavonyl-2,4-thiazolidinediones (flavonyl-2,4-TZD) (Ia-e, IIa-e and IIIa-g) were tested for their ability to inhibit rat kidney AR. Compound Ib showed the highest inhibitory activity (88.69 +/- 1.46%) whereas Ia, IIa, IIIa, IIIb also showed significant inhibitory activity (49.26 +/- 2.85, 67.29 +/- 1.09, 71.11 +/- 1.95, 64.86 +/- 1.21%, respectively).     

Structural and functional properties of aldose xylose reductase from the D-xylose-metabolizing yeast Candida tenuis

The primary structure of the aldose xylose reductase from Candida tenuis (CtAR) is shown to be 39% identical to that of human aldose reductase (hAR). The catalytic tetrad of hAR is completely conserved in CtAR (Tyr51, Lys80, Asp46, His113). The amino acid residues involved in binding of NADPH by hAR (D.K. Wilson, et al., Science 257 (1992) 81-84) are 64% identical in CtAR. Like hAR the yeast enzyme is specific for transferring the 4-pro-R hydrogen of the coenzyme. These properties suggest that CtAR is a member of the aldo/keto reductase superfamily. Unlike hAR the enzyme from C. tenuis has a dual coenzyme specificity and shows similar specificity constants for NADPH and NADH. It binds NADP(+) approximately 250 times less tightly than hAR. Typical turnover numbers for aldehyde reduction by CtAR (15-20 s(-1)) are up to 100-fold higher than corresponding values for hAR, probably reflecting an overall faster dissociation of NAD(P)(+) in the reaction catalyzed by the yeast enzyme.     

Structural bases for the inhibition of aldose reductase by phenolic compounds

Aldose reductase (ALR2) is an enzyme involved in the development of long-term diabetic complications. In the search for aldose reductase inhibitors less acidic than carboxylic acids, phenolic compounds related to benzopyran-4-one and chalcone are particularly interesting because they possess good inhibitory properties. In order to investigate the similarities between these two classes of compounds and to provide a structural basis for their inhibition of ALR2, the existing structure-activity relationships were reconsidered. To this end, the acidity constants of a set of chalcones were measured and compared with those of benzopyran-4-one derivatives. Then, having established the relevant protonation state of these phenolics at physiological pH, a conformational analysis was performed on the most active benzopyran-4-one and chalcone derivatives and the results were compared with the crystal structures of some analogues. Finally, molecular docking of the most active chalcone into the ALR2 binding site was performed, and the structure of the enzyme-inhibitor complex was compared with that of the complex formed between ALR2 and a previously-obtained benzopyran-4-one derivative.     

A reaction mechanism for aldose reductase from lens

Sheys and Doughty, (Sheys, G.H. and Doughty, C.C. (1979) Biochim. Biophys. Acta 242, 523-531) suggested a model for Rhodotorula (yeast) aldose reductase (alditol:NADP+ 1-oxidoreductase, EC 1.1.1.21) which offered a unified explanation for changes in reversibility, reaction mechanism, and effects of multivalent anions as well as substrate activation. The present paper extends this model to lens aldose reductase, explaining its similarities to the reverse reaction in Rhodotorula in regard to its reaction mechanism, as well as multivalent anion effects of sulfate, pyrophosphate and NADPH (above 20 micro M) and also substrate activation with glyceraldehyde involving formation of an abortive complex (above 50 micro M). Activation of lens aldose reductase resulted with multivalent anions, due to increased V max and apparent Km values with increasing concentration of multivalent anions. The lens enzyme mechanism is similar to the reverse reaction mechanism for the Rhodotorula enzyme, being partially random in character, based on NADP+ inhibitor studies presented here. The binding of NADPH appears to occur at a basic center containing arginine and possibly histidine. Evidence of the participation of these residues at the active center is based on time-course inactivation protection studies using reagents specific for these residues.     

Redox activation of aldose reductase in the ischemic heart

Aldose reductase (AR) reduces cytotoxic aldehydes and glutathione conjugates of aldehydes derived from lipid peroxidation. Its inhibition has been shown to increase oxidative injury and abolish the late phase of ischemic preconditioning. However, the mechanisms by which ischemia regulates AR activity remain unclear. Herein, we report that rat hearts subjected to ischemia, in situ or ex vivo, display a 2-4-fold increase in AR activity. The AR activity was not further enhanced by reperfusion. Activation increased Vmax of the enzyme without affecting the Km and decreased the sensitivity of the enzyme to inhibition by sorbinil. Enzyme activation could be prevented by pretreating the hearts with the radical scavenging thiol, N-(2-mercaptoproprionyl)glycine or the superoxide dismutase mimetic, Tiron, or by treating homogenates with dithiothreitol. In vitro, the recombinant enzyme was activated upon treatment with H2O2 and the activated, but not the native enzyme, formed a covalent adduct with the sulfenic acid-specific reagent dimedone. The enzyme activity in the ischemic, but not the nonischemic heart homogenates was inhibited by dimedone. Separation of proteins from hearts subjected to coronary occlusion by two-dimensional electrophoresis and subsequent matrix-assisted laser desorption ionization time-of-flight/mass spectrometry analysis revealed the formation of sulfenic acids at Cys-298 and Cys-303. These data indicate that reactive oxygen species formed in the ischemic heart activate AR by modifying its cysteine residues to sulfenic acids.