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

Grimshaw, Charles E.; Shahbaz, M.; Putney, C. G.

doi:10.1021/bi00494a027

Cited by 79 publications

(53 citation statements)

References 25 publications

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“…Because the ''safety belt'' structure in aldose reductase covers the NADPH cofactor, a significant conformational change in the loop 7 region appears necessary for cofactor binding and release (9). The large movement of loop 7 required for the formation and dissociation of the NADPH⅐enzyme complex has also been thought to be responsible for the biphasic kinetics of NADPH binding and release in aldose reductase (25,26). On the basis of this structure (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…However, for 2,5-DKGR A, the association and dissociation of the cofactor are not expected to be accompanied by large backbone movements of loop 7. Consequently, cofactor binding is postulated not to display biphasic kinetics, as is the case with most other aldo-keto reductases (25,26). The aldo-keto reductases recognize four broad categories of substrates: aldehydes, ketones, monosaccharides, and steroids.…”

Section: Discussionmentioning

confidence: 99%

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Crystal structure of 2,5-diketo- d -gluconic acid reductase A complexed with NADPH at 2.1-Å resolution

Khurana

Powers

Anderson

et al. 1998

Proc. Natl. Acad. Sci. U.S.A.

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The three-dimensional structure of Corynebacterium 2,5-diketo-D-gluconic acid reductase A (2,5-DKGR A; EC 1.1.1.-), in complex with cofactor NADPH, has been solved by using x-ray crystallographic data to 2.1-Å resolution. This enzyme catalyzes stereospecific reduction of 2,5-diketo-D-gluconate (2,5-DKG) to 2-keto-L-gulonate. Thus the threedimensional structure has now been solved for a prokaryotic example of the aldo-keto reductase superfamily. The details of the binding of the NADPH cofactor help to explain why 2,5-DKGR exhibits lower binding affinity for cofactor than the related human aldose reductase does. Furthermore, changes in the local loop structure near the cofactor suggest that 2,5-DKGR will not exhibit the biphasic cofactor binding characteristics observed in aldose reductase. Although the crystal structure does not include substrate, the two ordered water molecules present within the substrate-binding pocket are postulated to provide positional landmarks for the substrate 5-keto and 4-hydroxyl groups. The structural basis for several previously described active-site mutants of 2,5-DKGR A is also proposed. Recent research efforts have described a novel approach to the synthesis of L-ascorbate (vitamin C) by using a genetically engineered microorganism that is capable of synthesizing 2,5-DKG from glucose and subsequently is transformed with the gene for 2,5-DKGR. These modifications create a microorganism capable of direct production of 2-keto-L-gulonate from D-glucose, and the gulonate can subsequently be converted into vitamin C. In economic terms, vitamin C is the single most important specialty chemical manufactured in the world. Understanding the structural determinants of specificity, catalysis, and stability for 2,5-DKGR A is of substantial commercial interest.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Crystal structure of 2,5-diketo- d -gluconic acid reductase A complexed with NADPH at 2.1-Å resolution

Khurana

Powers

Anderson

et al. 1998

Proc. Natl. Acad. Sci. U.S.A.

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“…This procedure was continued until the concentration of salt was less than 5 mM. K d values were then determined as described previously [24].…”

Section: Methodsmentioning

confidence: 99%

Probing the inhibitor‐binding site of aldose reductase with site‐directed mutagenesis

Hohman

El‐Kabbani

Malamas

et al. 1998

European Journal of Biochemistry

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Aldose reductase (AR) has been implicated in the etiology of the secondary complications of diabetes, and enzyme inhibitors have been proposed as therapeutic agents. While effectively preventing the development of diabetic complications in animals, results from clinical studies of AR inhibitors have been disappointing, possibly due to poor potency in man. To assist in the design of more potent and specific inhibitors, crystallographic studies have attempted to identify enzyme-inhibitor interactions. Resolution of crystal complexes has suggested that the inhibitors bind to the enzyme active site and are held in place through hydrogen bonding and van der Waals interactions formed within two hydrophobic pockets. To confirm and extend these findings we quantified inhibitor activity with single, site-directed, mutant, human AR enzymes in which the apolar active-site residues tryptophan 20, Ϫ79, Ϫ111 and phenylalanine 115 were replaced with alanine or tyrosine, decreasing the potential for van der Waals interactions.Consistent with molecular models, the inhibitory activity of Tolrestat, Sorbinil and Zopolrestat decreased 800Ϫ2000-fold when tested with the mutant enzyme in which Trp20 was replaced with alanine. Further, alanine substitution for Trp111 decreased Zopolrestat's activity 400-fold, while mutations to Trp79 and Phe115 had little effect on the activity of any of the inhibitors. The alanine mutation at Trp111 had no effect on Tolrestat's activity but decreased the activity of Sorbinil by about 1000-fold. These latter effects were unanticipated based on the number of non-bonded interactions between the inhibitors, Tolrestat and Sorbinil, and Trp20 and Trp111 that have been identified in the crystal structures. In spite of these unexpected findings, our results are consistent with the hypothesis that AR inhibitors occupy the enzyme active site and that hydrophobic interactions between the enzyme and inhibitor contribute to inhibitor binding stability.Keywords : site-directed mutagenesis ; human aldose reductase; enzyme inhibition; polyol pathway ; diabetes.The polyol pathway has been implicated in the etiology of the secondary complications of diabetes. There are two enzymes in this pathway. The first, aldose reductase (AR), catalyzes the reduction of glucose to sorbitol and the second, sorbitol dehydrogenase, catalyzes the oxidation of sorbitol to fructose. AR has a relatively low affinity for glucose and during conditions of normal glycemia (80Ϫ110 mg/dl), where cellular glucose rapidly becomes phosphorylated via hexokinase, little substrate enters the pathway. During hyperglycemia, however, the cellular level of glucose greatly increases in tissues where glucose entry is independent of insulin. In these tissues which include the lens, retina, kidney and peripheral nerves, all sites of diabetes-induced

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“…In the reduction of aldehyde (forward reaction), AR follows a sequential ordered kinetic mechanism in which the co-factor NADPH binds before the aldehyde substrate and NADP ϩ is discharged after release of the alcohol product (19,20). Due to tight binding affinity, most endogenous hAR is anticipated to exist as a complex with NADP(H) in vivo.…”

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confidence: 99%

The Role of Cys-298 in Aldose Reductase Function

Balendiran

Sawaya²,

Schwarz

et al. 2011

Journal of Biological Chemistry

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Diabetic tissues are enriched in an "activated" form of human aldose reductase (hAR), a NADPH-dependent oxidoreductase involved in sugar metabolism. Activated hAR has reduced sensitivity to potential anti-diabetes drugs. The C298S mutant of hAR reproduces many characteristics of activated hAR, although it differs from wild-type hAR only by the replacement of a single sulfur atom with oxygen. Isothermal titration calorimetry measurements revealed that the binding constant of NADPH to the C298S mutant is decreased by a factor of two, whereas that of NADP ؉ remains the same. The aldo-keto reductases constitute a superfamily of NADPH-dependent oxidoreductases (1) that catalyze the reduction of a wide variety of aldehydes and ketones to their corresponding alcohols. The aldo-keto reductase family member aldose reductase (AR) 2 is implicated in the pathogenesis of a variety of diabetic complications including retinopathy, neuropathy, nephropathy, and cardiovascular diseases (2-7). Hence, AR has long been studied as a target for the design of potent active site inhibitors for the treatment of diabetes (8).Human AR (hAR) exists in both a native and an activated form, the later of which poses a potential obstacle to the development of diabetes drugs. Kinetic studies of activated hAR revealed differences (typically increases) in K m and V max for aldehyde substrates and, more importantly from a physiological standpoint, a marked reduction in sensitivity to hAR inhibitors (e.g. 1000-fold increase in K i for sorbinil, an otherwise potent active site inhibitor) (9 -13). Because activated enzyme forms are generally less liable to inhibition by hAR inhibitors, the therapeutic effectiveness of such drugs would be compromised if a large fraction of the enzyme underwent conversion to this activated form as has been discovered in diabetic tissues (9).The nature of the activated hAR form is not clearly understood but may result from oxidation of one of the six cysteines in the enzyme. Of the six cysteines found in hAR, only Cys-298 is located in sufficient proximity to the active site to directly perturb both enzyme activity and inhibitor sensitivity. Also, Cys-298 is the predominant site of thiolation on hAR, suggesting that its oxidation could be a mechanism for triggering conversion of hAR to the activated form (14). Indeed, when complexed with NADP ϩ , hAR is less susceptible to modification by many agents, including glutathione, nitric oxide, and 4-hydroxy-2-nonenal (HNE). That is, of all the cysteine residues in hAR, only Cys-298 is sufficiently close to NADP ϩ to be shielded from modification. Chemical modification and mutagenesis studies further distinguish Cys-298 as an important regulatory site on hAR. Either activation or inactivation of hAR can be effected by chemical modification of Cys-298. For example, the addition of oxidized glutathione results in glutathiolation of 15) and renders the enzyme catalytically inactive (14), whereas dithiodiethanol also targets Cys-298 but leads to hAR activation (16). Furthermore, a...

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

Cited by 79 publications

References 25 publications

Crystal structure of 2,5-diketo- d -gluconic acid reductase A complexed with NADPH at 2.1-Å resolution

Crystal structure of 2,5-diketo- d -gluconic acid reductase A complexed with NADPH at 2.1-Å resolution

Probing the inhibitor‐binding site of aldose reductase with site‐directed mutagenesis

The Role of Cys-298 in Aldose Reductase Function

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