Human Aldose Reductase: pK of Tyrosine 48 Reveals the Preferred Ionization State for Catalysis and Inhibition

Abstract

Detailed analyses of the pH variation of kinetic parameters for the forward aldehyde reduction and reverse alcohol oxidation reactions mediated by recombinant human aldose reductase, for inhibitor binding, and for kinetic isotope effects on aldehyde reduction have revealed that the pK value for the active site acid-base catalyst group Tyr48 is quite sensitive to the oxidation state of the bound nucleotide (NADPH or NADP+) and to the presence or absence of the Cys298 sulfhydryl moiety. Thus, the Tyr48 residue of C298A mutant enzyme displays a pK value that ranges from 7.6 in the productive *E.NADP+ complex that binds and reacts with alcohols to 8.7 in the productive *E.NADPH complex that binds and reacts with aldehyde substrates. For wild-type enzyme, Tyr48 in the latter complex displays a lower pK value of about 8.25. Assignment of the pK values was facilitated by the recognition and quantitation of the degree of stickiness of several aldehyde substrates in the forward reaction. The unusual pH dependence for Valdehyde/Et and DValdehyde, which decrease roughly 20-fold through a wave and remain constant at high pH, respectively, is shown to arise from the pH-dependent decrease in the net rate of NADP+ release. The results described are fully consistent with the chemical mechanism for aldose reductase catalysis proposed previously (Bohren et al., 1994) and, furthermore, establish that binding of the spirohydantoin class of aldose reductase inhibitors, e.g., sorbinil, occurs via a reverse protonation scheme in which the ionized inhibitor binds preferentially to the *E.NADP+ complex with Tyr48 present as the protonated hydroxyl form. The latter finding allows us to propose a unified model for high-affinity aldose reductase inhibitor binding that focuses on the transition state-like nature of the *E-Tyr48-OH.NADP+.inhibitor- complex.

Catalytic mechanism and substrate selectivity of aldo-keto reductases: Insights from structure-function studies of Candida tenuis xylose reductase

“…Results of pH studies revealed a kinetically unperturbed pK a of 8.50 for the AKR2B5-NADH complex above which the reductase activity was lost (32,34). The ionizable group of AKR2B5 to which the pK a of 8.50 can be assigned is most probably Tyr-51, consistent with pK ato-group assignments in other AKRs, particularly aldose reductase (16,18,33). Interestingly, the same pK a was observed in the pH profile of log k cat /K for a Asp-46!Asn Lys-80!Ala double mutant of AKR2B5 which was designed to substitute the side chain of Lys-80 without altering the overall net charge of the active site (34).…”

Polyol pathway and modulation of ischemia-reperfusion injury in Type 2 diabetic BBZ rat hearts

“…The polyol pathway enzyme aldose reductase catalyzes the reduction of aldo sugars and other saturated and unsaturated aldehydes [16][17][18][19][20]. This enzyme constitutes the first and the rate limiting step of the polyol pathway.…”

Design and synthesis of pyridazinone-substituted benzenesulphonylurea derivatives as anti-hyperglycaemic agents and inhibitors of aldose reductase – an enzyme embroiled in diabetic complications

“…These compounds (III and V) well occupied in the receptor cavity and forms hydrogen bonds and hydrophobic interactions (Figures 4 and 5). The sulfonamide group of III and V is anchored into the anion-binding site formed by Ala299, Leu300 and Trp111 and forms hydrogen-bonding with Trp111, which is key residue in binding and catalysis 32,33 . The 1,2,3,4-tetrahydronaphthalene ring core gets tightly trapped in the hydrophobic pocket formed by Phe122, Leu300, Leu301, Leu124 and Val130.…”

“…It has been shown that there is an approximately 85% sequence similarity between rat lens and human aldose reductase (ALR2), while the proposed active sites of both enzymes are identical 32 . The performed assay was based on a spectrometric measurement, which is proven to be a reliable method 33 , with DL-glyceraldehyde as the substrate and NADPH as the cofactor. Quercetin, a known ARI was used as a positive control.…”