Purification and properties of aldose reductase and aldehyde reductase II from human erythrocyte

Aldose reductase (EC 1.1.1.21) and aldehyde reductase II (L-hexonate dehydrogenase, EC 1.1.1.2) have been purified to homogeneity from human erythrocytes by using ion-exchange chromatography, chromatofocusing, affinity chromatography, and Sephadex gel filtration. Both enzymes are monomeric, Mr 32,500, by the criteria of the Sephadex gel filtration and polyacrylamide slab gel electrophoresis under denaturing conditions. The isoelectric pH's for aldose reductase and aldehyde reductase II were determined to be 5.47 and 5.06, respectively. Substrate specificity studies showed that aldose reductase, besides catalyzing the reduction of various aldehydes such as propionaldehyde, pyridine-3-aldehyde and glyceraldehyde, utilizes aldo-sugars such as glucose and galactose. Aldehyde reductase II, however, did not use aldo-sugars as substrate. Aldose reductase activity is expressed with either NADH or NADPH as cofactors, whereas aldehyde reductase II can utilize only NADPH. The pH optima for aldose reductase and aldehyde reductase II are 6.2 and 7.0, respectively. Both enzymes are susceptible to the inhibition by p-hydroxymercuribenzoate and N-ethylmaleimide. They are also inhibited to varying degrees by aldose reductase inhibitors such as sorbinil, alrestatin, quercetrin, tetramethylene glutaric acid, and sodium phenobarbital. The presence of 0.4 M lithium sulfate in the assay mixture is essential for the full expression of aldose reductase activity whereas it completely inhibits aldehyde reductase II. Amino acid compositions and immunological studies further show that erythrocyte aldose reductase is similar to human and bovine lens aldose reductase, and that aldehyde reductase II is similar to human liver and brain aldehyde reductase II.     

Purification and characterization of aldose reductase and aldehyde reductase from human kidney.

Aldose reductase and aldehyde reductases have been purified to homogeneity from human kidney and have molecular weights of 32,000 and 40,000 and isoelectric pH 5.8 and 5.3, respectively. Aldose reductase, beside catalyzing the reduction of various aldehydes, reduces aldo-sugars, whereas aldehyde reductase, does not reduce aldo-sugars. Aldose reductase activity is expressed with either NADH or NADPH as cofactor, whereas aldehyde reductase utilizes only NADPH. Both enzymes are inhibited to varying degrees by aldose reductase inhibitors. Antibodies against bovine lens aldose reductase precipitated aldose reductase but not aldehyde reductase. The sequence of addition of the substrates to aldehyde reductase is ordered and to aldose reductase is random, whereas for both the enzymes the release of product is ordered with NADP released last.     

Purification and characterization of human testis aldose and aldehyde reductase

1. Aldose reductase and aldehyde reductase were purified to homogeneity from human testis. 2. The molecular weight of aldose reductase and aldehyde reductase were estimated to be 36,000 and 38,000 by SDS-PAGE, and the pI values of these enzymes were found to be 5.9 and 5.1 by chromatofocusing, respectively. 3. Aldose reductase had activity for aldo-sugars, whereas aldehyde reductase was virtually inactive for aldo-sugars. The Km values of aldose reductase for D-glucose, D-galactose and D-xylose were 57, 49 and 6.2 mM, respectively. Aldose reductase utilized both NADPH and NADH as coenzymes, whereas aldehyde reductase only NADPH. 4. Sulfate ion caused 3-fold activation of aldose reductase, but little for that of aldehyde reductase. 5. Sodium valproate inhibited significantly aldehyde reductase, but not aldose reductase. Aldose reductase was inhibited strongly by aldose reductase inhibitors being in clinical trials at concentrations of the order of 10(-7)-10(-9) M. Aldehyde reductase was also inhibited by these inhibitors, but its susceptibility was less than aldose reductase. 6. Reaction of aldose reductase with pyridoxal 5'-phosphate (PLP) resulted ca 2.5-fold activation, but aldehyde reductase did not cause the activation. PLP-treated aldose reductase has lost the susceptibility to aldose reductase inhibitor.     

Characterization of aldose reductase and aldehyde reductase from rat testis

Aldose reductase (alditol:NAD(P)+ 1-oxidoreductase, EC 1.1.1.21) and aldehyde reductase (alcohol:NADP+ oxidoreductase, EC 1.1.1.2) were purified to a homogeneity from rat testis. The molecular weights of aldose reductase and aldehyde reductase were estimated to be 38,000 and 41,000 by SDS-polyacrylamide gel electrophoresis, and the pI values of these enzymes were found to be 5.3 and 6.1 by chromatofocusing, respectively. Aldose reductase had activity for aldo-sugars such as xylose, glucose and galactose, whereas aldehyde reductase was virtually inactive for these aldo-sugars. The Km values of aldose reductase for aldo-sugars were relatively high. When a correction was made for the fraction of aldo-sugar present as the aldehyde form, which is the real substrate of the enzyme, the Km values were much lower. Aldose reductase utilized both NADPH and NADH as coenzyme, whereas aldehyde reductase utilized only NADPH. Aldose reductase was activated significantly by sulfate ion, while aldehyde reductase was little affected. Both enzymes were inhibited strongly by the known aldose reductase inhibitors. However, aldehyde reductase was in general less susceptible to these inhibitors when compared to aldose reductase. Both aldose reductase and aldehyde reductase treated with pyridoxal 5-phosphate have lost the susceptibility to aldose reductase inhibitor, suggesting that in these two enzymes aldose reductase inhibitor interacts with a lysine residue.     

The kinetic mechanism of human placental aldose reductase and aldehyde reductase II

The kinetic mechanism of NADPH-dependent aldehyde reductase II and aldose reductase, purified from human placenta, has been studied using L-glucuronate and DL-glyceraldehyde as their respective substrates. For aldehyde reductase II, the initial velocity and product inhibition studies (using NADP and gulonate) indicate that the enzyme reaction sequence is ordered with NADPH binding to the free enzyme and NADP being the last product to be released. Inhibition patterns using menadione (an analog of the aldehydic substrate) and ATP-ribose (an analog of NADPH) are also consistent with a compulsory ordered reaction sequence. Isotope effects of deuterium-substituted NADPH (NADPD) also corroborate the above reaction scheme and indicate that hydride transfer is not the sole rate-limiting step in the reaction sequence. For aldose reductase, initial velocity patterns, product, and dead-end inhibition studies indicate a random binding pattern of the substrates and an ordered release of product; the coenzyme is released last. A steady-state random mechanism is also consistent with deuterium isotope effects of NADPD on the reaction sequence catalyzed by this enzyme. However, the hydride transfer step seems to be more rate determining for aldose reductase than for aldehyde reductase II.     

Interrelationships among human aldo-keto reductase: immunochemical, kinetic and structural properties

We have propsed earlier a three gene loci model to explain the expression of the aldo-keto reductases in human tissues. According to this model, aldose reductase is a monomer of α subunits, aldehyde reductase I is a dimer of α, β subunits, and aldehyde reductase II is a monomer of δ subunits. Using immunoaffinity methods, we have isolated the subunits of aldehyde reductase I (α and β) and characterized them by immunocompetition studies. It is observed that the two subunits of aldehyde reductase I are weakly held together in the holoenzyme and can be dissociated under high ionic conditions. Aldose reductase (α subunits) was generated from human placenta and liver aldehyde reductase I by ammonium sulfate (80% saturation). The kinetic, structural and immunological properties of the generated aldose reductase are similar to the aldose reductase obtained from the human erythrocytes and bovine lens. The main characteristic of the generated enzyme is the requirement of Li₂SO₄(0.4 M) for the expression of maximum enzyme activity, and its K_m for glucose is less than 50 mM, whereas the parent enzyme, aldehyde reductase I, is completely inhibited by 0.4 M Li₂SO₄ and its K_m for glucose is more than 200 mM. The β subunits of aldehyde reductase I did not have enzyme activity but cross-reacted with anti-aldehyde reductase I antiserum. The β subunits hybridized with the α subunits of placenta aldehyde I, and aldose reductase purified from human brain and bovine lens. The hybridized enzyme had the characteristics properties of placenta aldehyde reductase I.     

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.     

Inactivation of carbonyl reductase from human brain by phenylglyoxal and 2,3-butanedione: a comparison with aldehyde reductase and aldose reductase

Aldehyde reductase (alcohol:NADP+ oxidoreductase, EC 1.1.1.2), aldose reductase (alditol:NAD(P)+ 1-oxidoreductase, EC 1.1.1.21) and carbonyl reductase (secondary-alcohol:NADP+ oxidoreductase, EC 1.1.1.184) constitute the enzyme family of the aldo-keto reductases, a classification based on similar physicochemical properties and substrate specificities. The present study was undertaken in order to obtain information about the structural relationships between the three enzymes. Treatment of human aldehyde and carbonyl reductase with phenylglyoxal and 2,3-butanedione caused a complete and irreversible loss of enzyme activity, the rate of loss being proportional to the concentration of the dicarbonyl reagents. The inactivation of aldehyde reductase followed pseudo-first-order kinetics, whereas carbonyl reductase showed a more complex behavior, consistent with protein modification cooperativity. NADP+ partially prevented the loss of activity of both enzymes, and an even better protection of aldehyde reductase was afforded by the combination of coenzyme and substrate. Aldose reductase was partially inactivated by phenylglyoxal, but insensitive to 2,3-butanedione. The degree of inactivation with respect to the phenylglyoxal concentration showed saturation behavior. NADP+ partially protected the enzyme at low phenylglyoxal concentrations (0.5 mM), but showed no effect at high concentrations (5 mM). These findings suggest the presence of an essential arginine residue in the substrate-binding domain of aldehyde reductase and the coenzyme-binding site of carbonyl reductase. The effect of phenylglyoxal on aldose reductase may be explained by the modification of a reactive thiol or lysine rather than an arginine residue.     

Aldose and aldehyde reductases in human tissues

Immunochemical characterizations of aldose reductase and aldehyde reductases I and II, partially purified by DEAE-cellulose (DE-52) column chromatography from human tissues, were carried out by immunotitration, using antisera raised against the homogenous preparations of human and bovine lens aldose reductase and human placenta aldehyde reductase I and aldehyde reductase II. Anti-aldose reductase antiserum cross-reacted with aldehyde reductase I, anti-aldehyde reductase I antiserum cross-reacted with aldose reductase and anti-aldehyde reductase II antiserum precipitated aldehyde reductase II, but did not cross-react with aldose reductase or aldehyde reductase I from all the tissues examined. DE-52 elution profiles, substrate specificity and immunochemical characterization indicate that aldose reductase is present in human aorta, brain, erythrocyte and muscle; aldehyde reductase I is present in human kidney, liver and placenta; and aldehyde reductase II is present in human brain, erythrocyte, kidney, liver, lung and placenta. Monospecific anti-α and anti-β antisera were purified from placenta anti-aldehyde reductase I antiserum, using immunoaffinity techniques. Anti-α antiserum precipitated both aldehyde reductase I and aldose reductase, whereas anti-β antibodies cross-reacted with only aldehyde reductase I. Based on these studies, a three gene loci model is proposed to explain the genetic interrelationships among these enzymes. Aldose reductase is a monomer of α subunits, aldehyde reductase I is a dimer of α and β subunits and aldehyde reductase II is a monomer of δ subunits.     

Purification and properties of aldehyde reductases from human placenta

Aldehyde reductases (alcohol: NADP⁺-oxidoreductases, EC 1.1.1.2) I and II from human placenta have been purified to homogeneity. Aldehyde reductase I, molecular weight about 74 000, is a dimer of two nonidentical subunits of molecular weigths of about 32 500 and 39 000, whereas aldehyde erductase II is a monomer of about 32 500. Aldehyde reductase I can be dissociated into subunits under high ionic concentrations. The isoelectric pH for aldehyde reductases I and II are 5.76 and 5.20, respectively. Amino acid compositions of the two enzymes are significantly different. Placenta aldehyde reductase I can utilize glucose with a lower affinity, whereas aldehyde reductase II is not capable to reducing aldo-sugars. Similarly, aldehyde reductase I does not catalyse the reduction of glucuronate while aldehyde reductase II has a high affinity for glucuronate. Both enzymes, however, exhibit strong affinity towards various other aldehydes such as glyceraldehyde, propionaldehyde, and pyridine-3-aldehyde. The pH optima for aldehyde reductases I and II are 6.0 and 7.0, respectively. Aldehyde reductaase I can use both NADH and NADPH as cofactors, whereas aldehyde reductase II activity is dependent on NADPH only. Both enzymes are susceptible to inhibition by sulfhydryl group reagents, aldose reductase inhibitors, lithium sulfate, and sodium chloride to varying degrees.     

Stable preparation of aldose reductase isoenzymes from human placenta

An efficient, large-scale purification has been achieved for two aldose reductase isoenzymes from human placenta in stable form. The procedure included ammonium sulfate fractionation (45-75%), followed by chromatographies on Matrex Red A, DE-52 cellulose, and Matrex Orange A. The preparations were stable for at least 3 months at 3 degrees C. IC50 values toward sorbinil were similar to those reported for crude or partially purified enzymes, indicating that they retained native structures during the purification steps. The molecular weights of purified GAR1 and GAR2, named according to their order of elution with a salt gradient from a Matrex Red A column, were 36,600 and 40,300, respectively. Kinetic studies indicate that GAR1 belongs to an aldose reductase (a low-Km form) and GAR2 to an aldehyde reductase (a high-Km form). GAR2, an aldehyde reductase, was also active in the reduction of D-glucose, with an apparent Km comparable to that of GAR1 but with a Vmax only 14% that of GAR1.     

On-bead combinatorial techniques for the identification of selective aldose reductase inhibitors

Aldose reductase (AKR1B1; ALR2; E.C. 1.1.1.21) is an NADPH-dependent carbonyl reductase which has long been associated with complications resulting from the elevated blood glucose often found in diabetics. The development of effective inhibitors has been plagued by lack of specificity which has led to side effects in clinical trials. To address this problem, a library of bead-immobilized compounds was screened against fluorescently labeled aldose reductase in the presence of fluorescently labeled aldehyde reductase, a non-target enzyme, to identify compounds which were aldose reductase specific. Picked beads were decoded via novel bifunctional bead mass spec-based techniques and kinetic analysis of the ten inhibitors which were identified using this protocol yielded IC50 values in the micromolar range. Most importantly, all of these compounds showed a preference for aldose reductase with selectivities as high as approximately 7500-fold. The most potent of these exhibited uncompetitive inhibition versus the carbonyl-containing substrate D/L-glyceraldehyde with a Ki of 1.16 microM.     

Interrelationships among human aldo-keto reductases: immunochemical, kinetic and structural properties

We have proposed earlier a three gene loci model to explain the expression of the aldo-keto reductases in human tissues. According to this model, aldose reductase is a monomer of alpha subunits, aldehyde reductase I is a dimer of alpha, beta subunits, and aldehyde reductase II is a monomer of delta subunits. Using immunoaffinity methods, we have isolated the subunits of aldehyde reductase I (alpha and beta) and characterized them by immunocompetition studies. It is observed that the two subunits of aldehyde reductase I are weakly held together in the holoenzyme and can be dissociated under high ionic conditions. Aldose reductase (alpha subunits) was generated from human placenta and liver aldehyde reductase I by ammonium sulfate (80% saturation). The kinetic, structural and immunological properties of the generated aldose reductase are similar to the aldose reductase obtained from the human erythrocytes and bovine lens. The main characteristic of the generated enzyme is the requirement of Li2SO4 (0.4 M) for the expression of maximum enzyme activity, and its Km for glucose is less than 50 mM, whereas the parent enzyme, aldehyde reductase I, is completely inhibited by 0.4 M Li2SO4 and its Km for glucose is more than 200 mM. The beta subunits of aldehyde reductase I did not have enzyme activity but cross-reacted with anti-aldehyde reductase I antiserum. The beta subunits hybridized with the alpha subunits of placenta aldehyde reductase I, and aldose reductase purified from human brain and bovine lens. The hybridized enzyme had the characteristic properties of placenta aldehyde reductase I.     

Designing of acyl sulphonamide based quinoxalinones as multifunctional aldose reductase inhibitors

A series of quinoxalinone scaffold-based acyl sulfonamides were designed as aldose reductase inhibitors and evaluated for aldose reductase (ALR2)/aldehyde reductase (ALR1) inhibition and antioxidation. Compounds 9b-g containing styryl side chains at C3-side exhibited good ALR2 inhibitory activity and selectivity. Of them, 9g demonstrated the most potent inhibitory activity with an IC₅₀ value of 0.100?μM, and also exhibited excellent antioxidant activity, even comparable to the typical antioxidant Trolox. Compounds 9 had higher lipid-water partition coefficients relative to the carboxylic acid compounds 8, indicating that they may have better lipophilicity and membrane permeability. Structure-activity relationship (SAR) studies found that acyl trifluoromethanesulfonamide group at N1 and the C3-dihydroxystyryl side chain were the key structure for improving the aldose reductase inhibitory activity and antioxidant activity.     

Enzymes in pancreatic islets that use NADP(H) as a cofactor including evidence for a plasma membrane aldehyde reductase.

Recent evidence of a pyruvate malate shuttle capable of transporting a large amount of NADPH equivalents out of mitochondria in pancreatic islets suggests that cytosolic NADP(H) plays a role in beta cell metabolism. To obtain clues about these processes the activities of several NADPH-utilizing enzymes were estimated in pancreatic islets. Low levels of pyrroquinolone quinone (PQQ) and low levels of enzyme activity that reduce PQQ were found in islets. Low activities of palmitoyl-CoA and stearoyl-CoA desaturases were also detected. Significant activities of glutathione reductase, aldose reductase (EC.1.1.1.21) and aldehyde reductase (EC.1.1.1.2) were present in islets. Potent inhibitors of aldehyde and aldose reductases inhibited neither glucose-induced insulin release nor glucose metabolism in islets indicating that these reductases are not directly involved in glucose-induced insulin reaction. Over 90% of aldose reductase plus aldehyde reductase enzyme activity was present in the cytosol. Kinetic and chromatographic studies indicated that 60-70% of this activity in cytosol was due to aldehyde reductase and the remainder due to aldose reductase. Aldehyde reductase-like enzyme activity, as well as aldose reductase immunoreactivity, was detected in rat islet plasma membrane fractions purified by a polyethylene glycol-Dextran gradient or by a sucrose gradient. This is interesting in view of the fact that voltage-gated potassium channel beta subunits that contain aldehyde and aldose reductase-like NADPH-binding motifs have been detected in plasma membrane fractions of islets [Receptors and Channels 7: 237-243, 2000] and suggests that NADPH might have a yet unknown function in regulating activity of these potassium channels. Reductases may be present in cytosol to protect the insulin cell from molecules that cause oxidative injury.     

The developmentally regulated P100/11E gene of Leishmania major shows homology to a superfamily of reductase genes

The life cycle transformation of the protozoan parasite Leishmania from promastigote to amastigote is accompanied by changes in the level of expression of a number of proteins whose function may be necessary for parasite survival in the sandfly vector or mammalian host. To genetically characterize these proteins, we have cloned and characterized cDNA sequences that vary in abundance during the life cycle of Leishmania major. One sequence (P100/11E) encodes a poly(A+) RNA whose abundance is markedly elevated in promastigotes of L. major. The DNA sequence of the P100/11E cDNA predicts an acidic polypeptide of Mr = 32,000 which shows 40-46% similarity to the superfamily of reductase proteins including 2,5-diketo-D-gluconic acid reductase, aldose reductase, aldehyde reductase, and rho-crystallin. The P100/11E sequence of L. major contains the IPKS motif located at the active site of both aldose and aldehyde reductases. The P100/11H sequence was expressed in Escherichia coli, and the purified polypeptide was used to raise rabbit antisera which detect a protein of Mr = 35,000 in promastigotes of L. major. These results provide direct genetic evidence that L. major expresses a sequence homologous to the reductase superfamily as a developmentally regulated gene product in promastigotes.     

Cloning and sequence determination of human placental aldose reductase gene

The human aldose reductase gene has been cloned by screening a human placental cDNA library with antibodies against bovine lens aldose reductase. The nucleotide sequence of the entire coding region has been determined. The deduced amino acid sequence indicates that the human enzyme is 84% identical to the bovine lens aldose reductase and 85% identical to the rat lens aldose reductase. It is also very similar to the human aldehyde reductase, the bovine prostaglandin F synthase, and to the European common frog rho-crystallin. The deduced amino acid sequence also indicates that maturation of aldose reductase involves removal of the N-terminal methionine.     

Biogenic aldehyde metabolism in rat brain. Differential sensitivity of aldehyde reductase isoenzymes to sodium valproate

The effects of inhibitors of aldehyde reductase (alcohol:NADP+ oxidoreductase, EC 1.1.1.2) on the formation of 3-methoxy-4-hydroxyphenethylene glycol from normetanephrine have been studied in rat brain homogenates. The reaction pathway was shown to be unaffected by several inhibitors of the major (high Km) form of aldehyde reductase such as sodium valproate. Two isoenzymes of aldehyde reductase have been separated and characterized from rat brain. The minor (low Km) isoenzyme is shown to be relatively insensitive to sodium valproate and exhibits a similar inhibitor-sensitivity profile to that obtained for methoxyhydroxyphenethylene glycol formation. The low Km isoenzyme is therefore implicated in catecholamine metabolism. The metabolism of succinic semialdehyde and xylose by rat brain cytosol has also been examined. Aldose metabolism may also be attributed to the action of the low Km reductase, but the existence of a separate succinic semialdehyde reductase is postulated. The possible roles of aldehyde reductases in brain metabolism and the relationship between these enzymes and aldose reductase (alditol:NADP+ 1-oxidoreductase, EC 1.1.1.21) are discussed.     

Localization, isolation and properties of three NADPH-dependent aldehyde reducing enzymes from dog kidney

Three kinds of NADPH-dependent aldehyde reducing enzymes were present in the dog kidney. Aldose reductase was located in the inner medulla region and aldehyde reductase in all regions of the renal cortex, outer medulla and inner medulla. In addition, a new reductase designated tentatively as high-Km aldose reductase, which was converted into an aldose reductase-like enzyme, was present in the inner medulla region of the kidney. Aldose reductase, aldehyde reductase and high-Km aldose reductase were purified to homogeneity from each region of the dog kidney. The molecular weight of aldose reductase was estimated to be 38,500 by SDS-polyacrylamide gel electrophoresis and the isoelectric point was found to be 5.7 by chromatofocusing. Aldose reductase had activity for aldo-sugars such as D-xylose, D-glucose and D-galactose as substrates and utilized both NADPH and NADH as coenzymes. Sulfate ions resulted in over 2-fold activation of aldose reductase. All aldehyde reductases from the three regions had the same properties. The molecular weights and isoelectric points of aldehyde reductases were 40,000 and 6.1, respectively. The aldehyde reductases were inactive for D-hexose, utilized only NADPH as coenzyme and were not affected by sulfate ions. High-Km aldose reductase had a molecular weight of 38,500 and an isoelectric point of 5.4. It had activity for aldo-sugars, but showed much higher Km and lower kcat/Km values than aldose reductase. Sulfate ions inhibited high-Km aldose reductase. It was converted into an aldose reductase-like enzyme by incubation in phosphate buffer at pH 7.0. The three kinds of enzymes were strongly inhibited by the known aldose reductase inhibitors. However, aldehyde reductase and high-Km aldose reductase were, in general, less susceptible than aldose reductase.     

Transient-state and steady-state kinetic studies of the mechanism of NADH-dependent aldehyde reduction catalyzed by xylose reductase from the yeast Candida tenuis

Microbial xylose reductase, a representative aldo-keto reductase of primary sugar metabolism, catalyzes the NAD(P)H-dependent reduction of D-xylose with a turnover number approximately 100 times that of human aldose reductase for the same reaction. To determine the mechanistic basis for that physiologically relevant difference and pinpoint features that are unique to the microbial enzyme among other aldo/keto reductases, we carried out stopped-flow studies with wild-type xylose reductase from the yeast Candida tenuis. Analysis of transient kinetic data for binding of NAD(+) and NADH, and reduction of D-xylose and oxidation of xylitol at pH 7.0 and 25 degrees C provided estimates of rate constants for the following mechanism: E + NADH right arrow over left arrow E.NADH right arrow over left arrow E.NADH + D-xylose right arrow over left arrow E.NADH.D-xylose right arrow over left arrow E.NAD(+).xylitol right arrow over left arrow E.NAD(+) right arrow over left arrow E.NAD(+) right arrow over left arrow E + NAD(+). The net rate constant of dissociation of NAD(+) is approximately 90% rate limiting for k(cat) of D-xylose reduction. It is controlled by the conformational change which precedes nucleotide release and whose rate constant of 40 s(-)(1) is 200 times that of completely rate-limiting E.NADP(+) --> E.NADP(+) step in aldehyde reduction catalyzed by human aldose reductase [Grimshaw, C. E., et al. (1995) Biochemistry 34, 14356-14365]. Hydride transfer from NADH occurs with a rate constant of approximately 170 s(-1). In reverse reaction, the E.NADH --> E.NADH step takes place with a rate constant of 15 s(-1), and the rate constant of ternary-complex interconversion (3.8 s(-1)) largely determines xylitol turnover (0.9 s(-1)). The bound-state equilibrium constant for C. tenuis xylose reductase is estimated to be approximately 45 (=170/3.8), thus greatly favoring aldehyde reduction. Formation of productive complexes, E.NAD(+) and E.NADH, leads to a 7- and 9-fold decrease of dissociation constants of initial binary complexes, respectively, demonstrating that 12-fold differential binding of NADH (K(i) = 16 microM) vs NAD(+) (K(i) = 195 microM) chiefly reflects difference in stabilities of E.NADH and E.NAD(+). Primary deuterium isotope effects on k(cat) and k(cat)/K(xylose) were, respectively, 1.55 +/- 0.09 and 2.09 +/- 0.31 in H(2)O, and 1.26 +/- 0.06 and 1.58 +/- 0.17 in D(2)O. No deuterium solvent isotope effect on k(cat)/K(xylose) was observed. When deuteration of coenzyme selectively slowed the hydride transfer step, (D)()2(O)(k(cat)/K(xylose)) was inverse (0.89 +/- 0.14). The isotope effect data suggest a chemical mechanism of carbonyl reduction by xylose reductase in which transfer of hydride ion is a partially rate-limiting step and precedes the proton-transfer step.