人脑醛糖还原酶的纯化和一些基本性质

使用DEAE纤维素柱层析、PBE-94层析聚焦、NADP~+-Sepharose 4B亲合层析及SephadexG-100凝胶过滤分离纯化了人脑醛糖还原酶。在DEAE层析中,用咪唑-HCI缓冲液替代了磷酸缓冲液,改善了分离效果。在聚丙烯酰胺及SDS聚丙烯酰胺凝胶电泳中,纯化的人脑醛糖还原酶均呈一条区带。它的pI为5.6,最适pH为6.5,分子量为36,000,底物特异性和氨基酸组成与其它哺乳动物的醛糖还原酶有相似性。开链式醛糖是醛糖还原酶的真正底物,它在开链式和半缩醛的平衡体系中占比例极小,因而推知醛糖还原酶对此底物有很高的K_(cat)和K_(cat)/K_m值,能有效地将它们还原成相应的醇。     

Bovine lens aldose reductase: tight binding of the pyridine coenzyme

Analysis by HPLC of the protein-free supernatant obtained after denaturation of aldose reductase shows that the native form of the enzyme (ARb) contains a tightly bound NADP+, which is absent in the oxidatively modified form (ARa). The absorption, fluorescence, and circular dichroism spectra of ARb and ARa are consistent with the presence of the cofactor only in the native form of aldose reductase. On the other hand, the modified enzyme, in appropriate thiol reducing conditions, can tightly bind NADP+. This indicates a potential reversibility of the modification of aldose reductase, at least in terms of retention of the cofactor.     

Aldose reductase from human skeletal and heart muscle. Interconvertible forms related by thiol-disulfide exchange

Aldose reductase was purified from human skeletal and heart muscle by a rapid and efficient scheme involving Red Sepharose chromatography, chromatofocusing on Pharmacia PBE 94, and hydroxylapatite high pressure liquid chromatography. The scheme afforded homogeneous enzyme, 65% recovery, in 2 days. All muscle samples express aldose reductase but not the closely related aldehyde reductase. Aldose reductase is isolated in one of two forms that are distinguishable by their kinetic patterns with glyceraldehyde as substrate and which are interconvertible by treatment with dithiothreitol. Both forms are capable of catalyzing the reduction of glucose (Km = 68 mM), and both are highly sensitive to inhibition by aldose reductase inhibitors. The reduction of glucose was shown to be nearly stoichiometric with production of sorbitol (92 +/- 2%). Dialysis of aldose reductase in the absence of thiols or NADP converts it into a form that shows markedly different kinetic properties, including very weak catalytic activity toward glucose and insensitivity to aldose reductase inhibitors. This modified form can be converted back into the native form by dithiothreitol. Thiol titration of the two forms of aldose reductase with Ellman's reagent indicated that two thiol groups were lost when the enzyme was dialyzed in the absence of dithiothreitol or NADP.     

Aldose reductase from human psoas muscle. Affinity labeling of an active site lysine by pyridoxal 5'-phosphate and pyridoxal 5'-diphospho-5'-adenosine

The reaction of aldose reductase from human psoas muscle with either pyridoxal 5'-phosphate (PLP) or pyridoxal 5'-diphospho-5'-adenosine (PLP-AMP) results in a pseudo first-order 2-fold activation of the enzyme with the stoichiometric incorporation of 1 mol of either reagent per mol of enzyme. However, in addition to an increase in Vmax there was also an increase in Km for both substrate, DL-glyceraldehyde, and coenzyme, NADPH. This resulted in an overall decrease in catalytic efficiency (kcat/Km). Spectral analysis indicated that activation by both PLP and PLP-AMP was accompanied by Schiff's base formation and epsilon-pyridoxyllysine was identified in hydrolysates of the reduced enzyme PLP-complex. Digestion of either PLP-modified or PLP-AMP-modified aldose reductase with endoproteinase Lys-C followed by high performance liquid chromatography purification and amino acid sequencing of the pyridoxyllated peptide revealed that PLP and PLP-AMP had modified the same lysine residue. A 32-residue peptide containing the essential lysine was found to be highly homologous with a segment of the sequence of both human liver aldehyde reductase and rat lens aldose reductase. A tetrapeptide (Ile-Pro-Lys-Ser) containing the essential lysine was identical in all three enzymes. These results highlight the close structural similarity between members of the aldehyde reductase family.     

Expression of human aldose and aldehyde reductases. Site-directed mutagenesis of a critical lysine 262.

Human aldose reductase (EC 1.1.1.21) and aldehyde reductase (EC 1.1.1.2) are implicated in the development of diabetic complications by a variety of mechanisms, and a number of drugs to inhibit these enzymes have been proposed for the therapy and prevention of these complications. To probe the structure and function of these two enzymes, we used site-directed mutagenesis in the cDNAs of both enzymes to replace lysine 262 with methionine. Wild-type and mutant enzymes were overexpressed in Escherichia coli and purified by anion exchange and affinity chromatography. N-terminal sequence analysis, Western blots, and kinetic studies confirmed the identity of the recombinant wild-type enzymes with the native human placental and liver enzymes. Recombinant aldose reductase (hAR) and aldehyde reductase (hGR) have apparent kinetic constants virtually identical to their respective native enzymes. The mutant aldose reductase (hARK262 greater than M) shows a 66-fold increase in Km for NADPH with respect to the wild type (1.9 +/- 0.4 microM versus 125 +/- 14 microM), whereas the Km for DL-glyceraldehyde increased 35-fold (20 +/- 2 versus 693 +/- 41 microM). The same constants for the mutant aldehyde reductase (hGRK262 greater than M) increased 97- and 86-fold, respectively (from 2.0 +/- 0.4 to 194 +/- 16 microM and from 1.6 +/- 0.4 to 137 +/- 3 mM). These results indicate that lysine 262 in aldose reductase and aldehyde reductase is crucial to their catalytic activity by affecting co-factor binding.     

Aldose reductase: physiological role, properties and prospects for regulating activity]

Some properties of aldose reductase isolated from various sources and possible ways of regulation of the enzyme catalytic activity are reviewed. Mammalian aldose reductases are monomeric enzymes with M(r) of 30-40 kDa and a broad substrate specificity towards aldoses. The physiological role of this enzyme consists, apparently, in providing an additional pathway for utilization of glucose and removing toxic compounds carrying an aldehyde group from the cell. Aldose reductase is thought to play a key role in various hyperglycemic states, including diabetic cataract. The kinetics of the aldose reductase reaction is hyperbolic with NADPH and nonhyperbolic with glucose. The rate of the enzyme-catalyzed reaction is determined by the effector binding in the active of inhibitory center of the enzyme. Incubation with substrates leads to the activation of the enzyme which is accompanied by a decrease of the effector binding in the enzyme inhibitory center with a sharp decrease in the sensitivity of the activated enzyme to NADPH concentration changes in the presence of glucose excess. A mechanism underlying the catalytic effect of both native and activated forms of the enzyme is proposed.     

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.     

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.     

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.     

Purification and characterization of two aldose reductase isoenzymes from rabbit muscle

Using a modification of the procedure of Kormann et al. (Kormann, A. W., Hurst, R. O., and Flynn, T. G. (1972) Biochim. Biophys. Acta 258, 40-55) for the purification of glycerol dehydrogenase, two enzymes have been purified from the skeletal muscle of male rabbits. From a consideration of their properties these enzymes have been named aldose reductase 1 and aldose reductase 2, respectively. Both enzymes are monomeric by the criteria of gel filtration and polyacrylamide gel electrophoresis in sodium dodecyl sulfate and both reductases are immunologically identical as shown by double immunodiffusion and rocket immunoelectrophoresis. Aldose reductases 1 and 2 have almost identical amino acid compositions, their NH2 termini are blocked and the COOH termini of both enzymes are apparently identical. The enzymes differ, however, in molecular weight with aldose reductase 2 having Mr = 41,500 and aldose reductase 1 Mr 40,200. Both enzymes have the broad substrate specificity typical of the aldehyde reductase family of enzymes; Km values of aldose reductase 1 for aldo sugars were similar to those reported for rabbit lens aldose reductase, and both aldose reductase 1 and 2 were inhibited by the commercial aldose reductase inhibitors Alrestatin and Sorbinil. Two aldose reductases, immunologically and electrophoretically identical to the muscle enzymes, were found in rabbit lens. Two aldose reductases were also detected in the skeletal muscle of male rats and pigs and in pig and bovine lens. The presence of relatively large amounts of aldose reductase in muscle identifies a new and rich source of the enzyme.     

Chromatographic separation of activated and unactivated forms of aldose reductase

Chromatography of bovine kidney aldose reductase using Matrex Orange A affinity gel results in the separation of the unactivated and activated enzyme forms. The former washes through the column, while the latter is eluted with an NADPH step-gradient. The separated enzyme forms display Vmax and Km glycolaldehyde values, and relative sensitivities to inhibition by the aldose reductase inhibitor AL-1576 (spiro[2,7-difluorofluorene-9,4'-imidazolidine]-2',5'- dione), that are similar to those reported previously for the individual forms. However, because Vmax is 17-fold lower for the unactivated enzyme, the purification of aldose reductase via NADP(H) elution from a dye-ligand affinity matrix can result in the selective purification of only the activated enzyme form. These results have direct implications for the study of potential aldose reductase inhibitors, and may explain why linear double-reciprocal plots are commonly observed for enzyme prepared in this manner, while nonlinear plots are seen in other cases.     

Purification and partial characterization of an aldo-keto reductase from Saccharomyces cerevisiae.

A cytosolic aldo-keto reductase was purified from Saccharomyces cerevisiae ATCC 26602 to homogeneity by affinity chromatography, chromatofocusing, and hydroxylapatite chromatography. The relative molecular weights of the aldo-keto reductase as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and size exclusion chromatography were 36,800 and 35,000, respectively, indicating that the enzyme is monomeric. Amino acid composition and N-terminal sequence analysis revealed that the enzyme is closely related to the aldose reductases of xylose-fermenting yeasts and mammalian tissues. The enzyme was apparently immunologically unrelated to the aldose reductases of other xylose-fermenting yeasts. The aldo-keto reductase is NADPH specific and catalyzes the reduction of a variety of aldehydes. The best substrate for the enzyme is the aromatic aldehyde p-nitrobenzaldehyde (Km = 46 microM; kcat/Km = 52,100 s-1 M-1), whereas among the aldoses, DL-glyceraldehyde was the preferred substrate (Km = 1.44 mM; kcat/Km = 1,790 s-1 M-1). The enzyme failed to catalyze the reduction of menadione and p-benzoquinone, substrates for carbonyl reductase. The enzyme was inhibited only slightly by 2 mM sodium valproate and was activated by pyridoxal 5'-phosphate. The optimum pH of the enzyme is 5. These data indicate that the S. cerevisiae aldo-keto reductase is a monomeric NADPH-specific reductase with strong similarities to the aldose reductases.     

Purification and characterization of the recombinant human aldose reductase expressed in baculovirus system

Large quantities of recombinant human aldose reductase were produced using Spodoptera frugiperda cells and properties of the enzyme were characterized. Direct purification of the recombinant aldose reductase by affinity column chromatography using Matrex gel orange A yielded a single 36 kDa band, similar in size to the purified human muscle aldose reductase, on a sodium dodecyl sulfate-polyacrylamide gel after silver staining. The isoelectric point of the recombinant enzyme was 5.85 which is identical to the human muscle aldose reductase. Following the treatment with an acylamino-acid releasing enzyme, the blocked NH2-terminal amino acid was identified to be acetylalanine. The successive NH2-terminal sequence and that of the COOH-terminal peptide concurred with the expected translated sequence. Kinetic analyses of the recombinant enzyme activity for various substrates and the cofactor, NADPH, demonstrated a good agreement with the previously reported kinetic data on the purified human aldose reductase. A high concentration of (NH4)2SO4 elicited a significant increase in both Km and Kcat for DL-glyceraldehyde as well as D-glucose. Although IC50 values for most of the aldose reductase inhibitors with recombinant enzyme were found to fall within the comparable range of those obtained with nonhuman mammalian enzymes, the IC50 value for epalrestat was more than 10-fold higher in the recombinant enzyme. These results indicate that the recombinant human aldose reductase expressed in the baculovirus system possesses structurally and enzymatically similar properties as those reported for the native human enzyme and should serve as a superior enzyme preparation to nonhuman mammalian enzymes for the screening of the efficacy and potency of newly developed aldose reductase inhibitors.     

C-terminal cysteines of Tn501 mercuric ion reductase.

Mercuric ion reductase (MerA) catalyzes the reduction of Hg(II) to Hg(0) as the last step in the bacterial mercury detoxification pathway. A member of the flavin disulfide oxidoreductase family, MerA contains an FAD prosthetic group and redox-active disulfide in its active site. However, the presence of these two moieties is not sufficient for catalytic Hg(II) reduction, as other enzyme family members are potently inhibited by mercurials. We have previously identified a second pair of active site cysteines (Cys558 Cys559 in the Tn501 enzyme) unique to MerA, that are essential for high levels of mercuric ion reductase activity [Moore, M. J., & Walsh, C. T. (1989) Biochemistry 28, 1183; Miller, S. M., et al. (1989) Biochemistry 28, 1194]. In this paper, we have examined the individual roles of Cys558 and Cys559 by site-directed mutagenesis of each to alanine. Phenotypic analysis indicates that both merA mutations result in a total disruption of the Hg(II) detoxification pathway in vivo, while characterization of the purified mutant enzymes in vitro shows each to have differential effects on catalytic function. Compared to wild-type enzyme, the C558A mutant shows a 20-fold reduction in kcat and a 10-fold increase in Km, for an overall decrease in catalytic efficiency of 200-fold in kcat/Km. In contrast, mutation of Cys559 to alanine results in less than a 2-fold reduction in kcat and an increase in Km of only 4-5 fold for an overall decrease in catalytic efficiency of only ca. 10-fold in vitro. From these results, it appears that Cys558 plays a more important role in forming the reducible complex with Hg(II), while both Cys558 and Cys559 seem to be involved in efficient scavenging (i.e., tight binding) of Hg(II).     

Aldose reductase from human psoas muscle. Purification, substrate specificity, immunological characterization, and effect of drugs and inhibitors

Aldose reductase (ALR2) has been purified to homogeneity from human psoas muscle. From sodium dodecyl sulfate-polyacrylamide electrophoresis the enzyme is monomeric and has a molecular weight of 37,000. ALR2 catalyzes the primarily NADPH-dependent reduction of a wide variety of aldehydes, although the enzyme can also utilize NADH. The best substrates for ALR2 are aromatic aldehydes (e.g. pyridine-3-aldehyde; Km = 9 microM; kcat/Km = 150,000 s-1 M-1), while among aldoses DL-glyceraldehyde is the preferred substrate (Km = 72 microM; kcat/Km = 17,250). Low (100 microM) concentrations of CaCl2 and CaSO4 cause a marked inhibition (90%) of ALR2 as do higher concentrations (0.2 M) of MgCl2. (NH4)2SO4 caused a 2-fold activation of ALR2. The enzyme is also inhibited by quercetin and the commercially developed aldose reductase inhibitors alrestatin and sorbinil. ALR2 is inhibited only very slightly by sodium valproate and barbiturates. ALR2 cross-reacts immunologically with human brain and human placental aldose reductase and with ALR2 from monkey tissue. There is no precipitin cross-reaction of ALR2 with aldose reductases from other species nor with human aldehyde reductase 1 (ALR1) or with ALR1 from other species. The data show that human muscle is a new and relatively rich source of a monomeric NADPH/NADH reductase which is clearly identifiable as aldose reductase.     

The crystal structure of the aldose reductase.NADPH binary complex.

Aldose reductase is an NADPH-dependent oxidoreductase that catalyzes the reduction of a broad range of aldehydes, including glucose. Since aldose reductase has been strongly implicated in the development of the chronic complications of diabetes mellitus, much effort has been devoted to understanding the structure and mechanism of this enzyme, and many aldose reductase inhibitors have been developed as potential drugs for the treatment of these complications. We describe here the 2.75 A crystal structure of recombinant human aldose reductase (Cys-298 to Ser mutant) complexed with NADPH. This mutant displays unusual kinetic behavior characterized by high Km/high Vmax substrate kinetics and reduced sensitivity to certain aldose reductase inhibitors. The crystal structure revealed that the enzyme is a beta/alpha-barrel with the coenzyme-binding domain located at the carboxyl-terminal end of the parallel strands of the barrel. The enzyme undergoes a large conformational change upon binding NADPH which involves the reorientation of loop 7 to a position which appears to lock the coenzyme into place. NADPH is bound to aldose reductase in an unusual manner, more similar to FAD- rather than NAD(P)-dependent oxidoreductases. No disulfide bridges were observed in the crystal structure.     

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.     

Regulation of aldose reductase activity. Mechanism of action of an activated form of the enzyme]

Activation of bovine eye lens aldose reductase during its incubation with NADPH and glucose was studied. The activated form of the enzyme was isolated, and the rate of glucose reduction measured within a broad range of substrate concentrations. Spectrophotometric titration and equilibrium gel-filtration were used to study the interaction of the enzyme active center with substrates. It was found that the reaction kinetics obeys the mechanism of a quasi-equilibrium binding of substrates with isomerization of the enzyme complexes with nicotinamide dinucleotide phosphates. This activation is accompanied by a transition from non-ordered to highly ordered binding of the substrates. The effect of ligands in the catalytic and inhibitory centers of the activated enzyme on the catalytic reaction was examined. It was found that the activated form of aldose reductase is characterized by a lower affinity of the inhibitory center for the flavonoid, morin. Morin binding not only inhibits the reaction but also prevents the activation of the enzyme.     

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.     

Effects of conversion of an invariant tryptophan residue to phenylalanine on the function of human dihydrofolate reductase

The binding site residue Trp-24 is conserved in all vertebrate and bacterial dihydrofolate reductases of known sequence. To determine its effects on enzyme properties, a Trp-24 to Phe-24 mutant (W-24-F) of human dihydrofolate reductase has been constructed by oligodeoxynucleotide site-directed mutagenesis. The W-24-F mutant enzyme appears to have a more open or flexible conformation as compared to the wild-type human dihydrofolate reductase on the basis of results of a number of studies. These studies include competitive ELISA using peptide-specific antibodies against human dihydrofolate reductase, thermal stability, and protease susceptibility studies of both mutant W-24-F and wild-type enzymes. It is concluded that Trp-24 is important for maintaining the structural integrity of the native enzymes. Changes in relative fluorescence quantum yield indicate that Trp-24 is buried and its fluorescence quenched relative to the other two tryptophan residues in the wild-type human reductase. Kinetic studies indicate that kcat values for W-24-F are increased in the pH range of 4.5-8.5 with a 5-fold increase at pH 7.5 as compared to the wild-type enzyme. However, the catalytic efficiency of W-24-F decreases rapidly as the pH is increased from 7.5 to 9.5. The Km values for dihydrofolate are also increased for W-24-F in the pH range of 4.5-9.5 with a 30-fold increase at pH 7.5, while the Km value for NADPH increases only ca. 1.4-fold at pH 7.5 as compared to the wild type.(ABSTRACT TRUNCATED AT 250 WORDS)