Change in stereospecificity of bovine lens aldose reductase modified by oxidative stress

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.

Section: Discussioncontrasting

confidence: 57%

mentioning

confidence: 99%

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

Grimshaw¹,

Bohren²,

Lai³

et al. 1995

“…Thus, in the C298A mutant the conformational change shows a small effect, and the large effect is seen for aldehyde and ARI binding. Finally, for oxidized hAR, which the studies of Mura and co-workers (Del Corso et al, 1989Giannessi et al, 1993;Cappiello et al, 1994) suggest may consist of a mixed disulfide between Cys298 and an organic thiol such as cysteine or glutathione, there is apparently no effect on ku, but the binding of either DL-glyceraldehyde or Sorbinil is drastically worsened. The latter result suggests that a mixed disulfide formed at the Cys298 position may be able to interact directly with the active site in such a way that the closed conformation of the nucleotide enfolding loop is stabilized, but the binding of aldehyde or ARI in the catalytic site is essentially blocked.…”

Section: Discussionmentioning

confidence: 96%

“…residue in hAR, either by chemical (DelCorso et al, 1989;Liu et al, 1989Liu et al, , 1992aBhatnagar et al, 1989Bhatnagar et al, , 1994Giannessi et al, 1993;Cappiello et al, 1994) or molecular biological means(Petrash et al, , 1993Bohren & Gabbay, 1993), results in enzyme forms displaying altered kinetic properties. Comparison of the results in Table4with the corresponding results from the preceding paper(Grimshaw et al, 1995a) shows that substitution of Cys298 with an alanine residue results in a nearly 9-fold increase in the forward rate of D-xylose reduction, a 37-fold increase in Á"xyiose, and a small, but significant increase in the deuterium isotope effects on both Vxyiose and V/Kxyiose• By applying the pre-steady-state kinetic data reported here to the kinetic model, we can now rationalize each of these changes in terms of the individual rate constants.…”

mentioning

confidence: 99%

Human Aldose Reductase: Subtle Effects Revealed by Rapid Kinetic Studies of the C298A Mutant Enzyme

Grimshaw¹,

Bohren²,

Lai³

et al. 1995

Transient kinetic data for D-xylose reduction with NADPH and NADPD and for xylitol oxidation with NADP+ catalyzed by recombinant C298A mutant human aldose reductase at pH 8 have been used to obtain estimates for each of the rate constants in the complete reaction mechanism as outlined for the wild-type enzyme in the preceding paper (Grimshaw et al., 1995a). Analysis of the resulting kinetic model shows that the nearly 9-fold increase in Vxylose/Et for C298A mutant enzyme relative to wild-type human aldose reductase is due entirely to an 8.7-fold increase in the rate constant for the conformational change that converts the tight (Ki NADP+ = 0.14 microM) binary *E.NADP+ complex to the weak (Kd NADP+ = 6.8 microM) E.NADP+ complex from which NADP+ is released. Evaluation of the rate expressions derived from the kinetic model for the various steady-state kinetic parameters reveals that the 37-fold increase in Kxylose seen for C298A relative to wild-type aldose reductase is largely due to this same increase in the net rate of NADP+ release; the rate constant for xylose binding accounts for only a factor of 5.5. A similar 17-fold increase in the rate constant for the conformational change preceding NADPH release does not, however, result in any increase in Vxylitol/Et, because hydride transfer is largely rate-limiting for reaction in this direction.(ABSTRACT TRUNCATED AT 250 WORDS)

“…Although product versus time plots for ALR2-catalyzed reduction of glyceraldehyde display a time-dependent decay in rate similar to that seen for glycolaldehyde, spectroscopic evidence suggests that adduct formation with the three-carbon aldoses may be complicated by a second reaction that is dependent on the stereochemistry at the -carbon (Grimshaw, 1989). In addition, because the stereospecificity of ALR2 for the glyceraldehyde enantiomers appears to change under certain conditions (Del Corso et al, 1989;Grimshaw et al, 1989), we chose to study the simple two-carbon aldose, glycolaldehyde.…”

mentioning

confidence: 99%

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

Grimshaw¹,

Shahbaz²,

Putney³

1990

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.