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.     

Identification of Pig Brain Aldehyde Reductases with the High-Km Aldehyde Reductase, the Low-Km Aldehyde Reductase and Aldose Reductase, Carbonyl Reductase, and Succinic Semialdehyde Reductase

Four NADPH-dependent aldehyde reductases (ALRs) isolated from pig brain have been characterized with respect to substrate specificity, inhibition by drugs, and immunological criteria. The major enzyme, ALR1, is identical in these respects with the high-Km aldehyde reductase, glucuronate reductase, and tissue-specific, e.g., pig kidney aldehyde reductase. A second enzyme, ALR2, is identical with the low-Km aldehyde reductase and aldose reductase. The third enzyme, ALR3, is carbonyl reductase and has several features in common with prostaglandin-9-ketoreductase and xenobiotic ketoreductase. The fourth enzyme, unlike the other three which are monomeric, is a dimeric succinic semialdehyde reductase. All four of these enzymes are capable of reducing aldehydes derived from the biogenic amines. However, from a consideration of their substrate specificities and the relevant Km and Vmax values, it is likely that it is ALR2 which plays a primary role in biogenic aldehyde metabolism. Both ALR1 and ALR2 may be involved in the reduction of isocorticosteroids. Despite its capacity to reduce ketones, ALR3 is primarily an aldehyde reductase, but clues as to its physiological role in brain cannot be discerned from its substrate specificity. The capacity of succinic semialdehyde reductase to reduce succinic semialdehyde better than any other substrate shows that this reductase is aptly named and suggests that its primary role is the maintenance in brain of physiological levels of gamma-hydroxybutyrate.     

Aldehyde and aldose reductases from human placenta. Heterogeneous expression of multiple enzyme forms

Aldehyde reductase (ALR1) and aldose reductase (ALR2) were purified from human placenta by a rapid and efficient scheme that included rapid extraction of both reductases from 100,000 x g supernatant material with Red Sepharose followed by purification by chromatofocusing on Pharmacia PBE 94 and then chromatography on a hydroxylapatite high performance liquid chromatography column. Expression of ALR1 and ALR2 in placenta is variable with ALR1/ALR2 ratios ranging from 1:4 to 4:1. ALR1 and ALR2 are immunochemically distinct. ALR1 shows broad specificity for aldehydes but does not efficiently catalyze the reduction of glucose due to poor binding (Km = 2.5 M). ALR1 exhibits substrate inhibition with many substrates. ALR2 also shows broad specificity for aldehydes. Although glucose is a poor substrate for ALR2 compared with other substrates, the affinity of ALR2 for glucose (Km = 70 mM) suggests that glucose can be a substrate under hyperglycemic conditions. ALR2 shows normal hyperbolic kinetics with most substrates except with glyceraldehyde, which exhibits substrate activation. Treatment of ALR2 with dithiothreitol converted it into a form that exhibited hyperbolic kinetics with glyceraldehyde. Dithiothreitol treatment of ALR2 did not alter its properties toward other substrates or affect its inhibition by aldose reductase inhibitors such as sorbinil (2,4-dihydro-6-fluorospiro-[4H-1-benzopyran-4,4'-imidazolidine]-2' ,5'- dione), tolrestat (N-[[6-methoxy-5-(trifluoromethyl)-1-naphthalenyl]thioxomethyl]-N- methylglycine), or statil (3-[(4-bromo-2-fluorophenyl)methyl]-3,4-dihydro-4-oxo-1-phthalazineac etic acid).     

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.     

Involvement of cysteine residues in catalysis and inhibition of human aldose reductase. Site-directed mutagenesis of Cys-80, -298, and -303.

In order to study the potential role of cysteinyl residues in catalysis and inhibition of human aldose reductase, mutants containing cysteine to serine substitution at positions 80 (ALR2:C80S), 298 (ALR2:C298S), and 303 (ALR2:C303S) were constructed. Mutation of Cys298 resulted in the most profound changes, as ALR2:C298S displayed 4- to 5-fold elevation in K'm(NADPH), K'm(DL-glyceraldehyde), and kcat(DL-glyceraldehyde) relative to wild type aldose reductase as well as a 10-fold higher Ki for the aldose reductase inhibitor sorbinil. Wild type and mutant reductases were equally sensitive to tolrestat, a structurally different reductase inhibitor. Carboxymethylation of the wild type enzyme or the C80S and C303S mutants led to a modest decrease in kcat as well as an increase in K'm(DL-glyceraldehyde) and Ki(sorbinil). These parameters were not significantly changed when ALR2:C298S was subjected to carboxymethylation. Lithium sulfate caused activation of ALR2:WT, C80S, and C303S but did not significantly affect the activity of ALR2:C298S. The differential sensitivity of wild type and mutant reductases to inhibition by sorbinil and tolrestat, before and after carboxymethylation, indicates that these inhibitors bind at different sites. These results suggest that Cys-298 is present near the active site and constitutes a regulatory group which controls the catalytic activity and inhibitor sensitivity of the enzyme.     

Reduction of trioses by NADPH-dependent aldo-keto reductases. Aldose reductase, methylglyoxal, and diabetic complications.

The substrate specificities of human aldose reductase and aldehyde reductase toward trioses, triose phosphates, and related three-carbon aldehydes and ketones were evaluated. Both enzymes are able to catalyze the NADPH-dependent reduction of all of the substrates used. Aldose reductase shows more discrimination among substrates than does aldehyde reductase and is generally the more efficient catalyst. The best substrate for aldose reductase is methylglyoxal (kcat = 142 min-1, kcat/Km = 1.8 x 10(7) M-1 min-1), a toxic 2-oxo-aldehyde that is produced nonenzymatically from triose phosphates and enzymatically from acetone/acetol metabolism. D- and L-glyceraldehyde and D- and L-lactaldehyde are also good substrates for aldose reductase. The aldose reductase-catalyzed reduction of methylglyoxal produces 95% acetol, 5% D-lactaldehyde. Further reduction of acetol produces only L-1,2-propanediol. Acetol and propanediol are two products that accumulate in uncontrolled diabetes. Both acetol and methylglyoxal were compared with glucose for their abilities to produce covalent modification of albumin. All three of these carbonyl compounds reacted with albumin to produce modified proteins with new absorption and emission bands that are spectrally similar. Both methylglyoxal and acetol are much more reactive than glucose. A new integrative model of diabetic complications is proposed that combines the aldose reductase/polyol pathway theory and the nonenzymatic glycation theory except that emphasis is placed both on methylglyoxal/acetol metabolism and on glucose metabolism.     

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 a novel erythrose reductase from Candida magnoliae

Erythritol biosynthesis is catalyzed by erythrose reductase, which converts erythrose to erythritol. Erythrose reductase, however, has never been characterized in terms of amino acid sequence and kinetics. In this study, NAD(P)H-dependent erythrose reductase was purified to homogeneity from Candida magnoliae KFCC 11023 by ion exchange, gel filtration, affinity chromatography, and preparative electrophoresis. The molecular weights of erythrose reductase determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration chromatography were 38,800 and 79,000, respectively, suggesting that the enzyme is homodimeric. Partial amino acid sequence analysis indicates that the enzyme is closely related to other yeast aldose reductases. C. magnoliae erythrose reductase catalyzes the reduction of various aldehydes. Among aldoses, erythrose was the preferred substrate (K(m) = 7.9 mM; k(cat)/K(m) = 0.73 mM(-1) s(-1)). This enzyme had a dual coenzyme specificity with greater catalytic efficiency with NADH (k(cat)/K(m) = 450 mM(-1) s(-1)) than with NADPH (k(cat)/K(m) = 5.5 mM(-1) s(-1)), unlike previously characterized aldose reductases, and is specific for transferring the 4-pro-R hydrogen of NADH, which is typical of members of the aldo/keto reductase superfamily. Initial velocity and product inhibition studies are consistent with the hypothesis that the reduction proceeds via a sequential ordered mechanism. The enzyme required sulfhydryl compounds for optimal activity and was strongly inhibited by Cu(2+) and quercetin, a strong aldose reductase inhibitor, but was not inhibited by aldehyde reductase inhibitors and did not catalyze the reduction of the substrates for carbonyl reductase. These data indicate that the C. magnoliae erythrose reductase is an NAD(P)H-dependent homodimeric aldose reductase with an unusual dual coenzyme specificity.     

Purification and properties of low-Km aldehyde reductase from ox brain

A low-Km aldehyde reductase (alcohol:NADP+ oxidoreductase, EC 1.1.1.2), which may be identical with aldose reductase (alditol:NADP+ 1-oxidoreductase, EC 1.1.1.21), has been purified from ox brain to homogeneity. It was shown to be a monomer with Mr values of 31 000 and 35 100 being obtained by gel filtration and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate, respectively. The enzyme catalyses the NADPH-dependent reduction of a number of aromatic and sugar aldehydes. The activity of the enzyme with 133 microM NADH was about one-third of that with 120 microM NADPH. Activity with both these coenzymes was optimum at pH 6.2 and was inhibited by increasing the ionic strength with KCl, NaCl or NaNO3. In contrast, the activity was stimulated by sodium phosphate. The activity with NADH as the coenzyme was more sensitive to stimulation by phosphate and to inhibition by increasing ionic strength than that determined with NADPH.     

Acryloyl-CoA reductase from Clostridium propionicum. An enzyme complex of propionyl-CoA dehydrogenase and electron-transferring flavoprotein.

Acryloyl-CoA reductase from Clostridium propionicum catalyses the irreversible NADH-dependent formation of propionyl-CoA from acryloyl-CoA. Purification yielded a heterohexadecameric yellow-greenish enzyme complex [(alpha2betagamma)4; molecular mass 600 +/- 50 kDa] composed of a propionyl-CoA dehydrogenase (alpha2, 2 x 40 kDa) and an electron-transferring flavoprotein (ETF; beta, 38 kDa; gamma, 29 kDa). A flavin content (90% FAD and 10% FMN) of 2.4 mol per alpha2betagamma subcomplex (149 kDa) was determined. A substrate alternative to acryloyl-CoA (Km = 2 +/- 1 microm; kcat = 4.5 s-1 at 100 microm NADH) is 3-buten-2-one (methyl vinyl ketone; Km = 1800 microm; kcat = 29 s-1 at 300 microm NADH). The enzyme complex exhibits acyl-CoA dehydrogenase activity with propionyl-CoA (Km = 50 microm; kcat = 2.0 s-1) or butyryl-CoA (Km = 100 microm; kcat = 3.5 s-1) as electron donor and 200 microm ferricenium hexafluorophosphate as acceptor. The enzyme also catalysed the oxidation of NADH by iodonitrosotetrazolium chloride (diaphorase activity) or by air, which led to the formation of H2O2 (NADH oxidase activity). The N-terminus of the dimeric propionyl-CoA dehydrogenase subunit is similar to those of butyryl-CoA dehydrogenases from several clostridia and related anaerobes (up to 55% sequence identity). The N-termini of the beta and gamma subunits share 40% and 35% sequence identities with those of the A and B subunits of the ETF from Megasphaera elsdenii, respectively, and up to 60% with those of putative ETFs from other anaerobes. Acryloyl-CoA reductase from C. propionicum has been characterized as a soluble enzyme, with kinetic properties perfectly adapted to the requirements of the organism. The enzyme appears not to be involved in anaerobic respiration with NADH or reduced ferredoxin as electron donors. There is no relationship to the trans-2-enoyl-CoA reductases from various organisms or the recently described acryloyl-CoA reductase activity of propionyl-CoA synthase from Chloroflexus aurantiacus.     

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.     

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.     

Characterization and nucleotide binding properties of a mutant dihydropteridine reductase containing an aspartate 37-isoleucine replacement.

Kinetic constants for the interaction of NADH and NADPH with native rat dihydropteridine reductase (DHPR) and an Escherichia coli expressed mutant (D-37-I) have been determined. Comparison of kcat and Km values measured employing quinonoid 6,7-dimethyldihydropteridine (q-PtH2) as substrate indicate that the native enzyme has a considerable preference for NADH with an optimum kcat/Km of 12 microM-1 s-1 compared with a figure of 0.25 microM-1 s-1 for NADPH. Although the mutant enzyme still displays an apparent preference for NADH (kcat/Km = 1.2 microM-1 s-1) compared with NADPH (kcat/Km = 0.6 microM-1 s-1), kinetic analysis indicates that NADH and NADPH have comparable stickiness in the D-37-I mutant. The dihydropteridine site is less affected, since the Km for q-PtH2 and K(is) for aminopterin are unchanged and the 14-26-fold synergy seen for aminopterin binding to E.NAD(P)H versus free E is decreased by less than 2-fold in the D-37-I mutant. No significant changes in log kcat and log kcat/Km versus pH profiles for NADH and NADPH were seen for the D-37-I mutant enzyme. However, the mutant enzyme is less stable to proteolytic degradation, to elevated temperature, and to increasing concentrations of urea and salt than the wild type. NADPH provides maximal protection against inactivation in all cases for both the native and D-37-I mutant enzymes. Examination of the rat DHPR sequence shows a typical dinucleotide binding fold with Asp-37 located precisely in the position predicted for the acidic residue that participates in hydrogen bond formation with the 2'-hydroxyl moiety of all known NAD-dependent dehydrogenases. This assignment is consistent with x-ray crystallographic results that localize the aspartate 37 carboxyl within ideal hydrogen bonding distance of the 2'- and 3'-hydroxyl moieties of adenosine ribose in the binary E.NADH complex.     

Human carboxypeptidase M. Purification and characterization of a membrane-bound carboxypeptidase that cleaves peptide hormones

A membrane-bound neutral carboxypeptidase B-like enzyme was solubilized from human placental microvilli with 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS) and purified to homogeneity by ion-exchange chromatography and affinity chromatography on arginine-Sepharose. It gave a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent Mr of 62,000 with or without reduction. The enzyme is a glycoprotein as shown by its high affinity for concanavalin A-Sepharose and reduction in mass to 47,600 daltons after chemical deglycosylation. It has a neutral pH optimum, is activated by CoCl2, and inhibited by o-phenanthroline, 2-mercaptomethyl-3-guanidinoethylthiopropanoic acid, or cadmium acetate, indicating it is a metallopeptidase. The enzyme cleaves arginine or lysine from the COOH terminus of synthetic peptides (e.g. Bz-Gly-Arg, Bz-Gly-Lys, Bz-Ala-Lys, dansyl-Ala-Arg, where Bz is benzoyl and dansyl is 5-dimethylaminonaphthalene-1-sulfonyl) as well as from several biologically active substrates: dynorphin A(1-13), Met5-Arg6-enkephalin (Km = 46 microM, kcat = 934 min-1), bradykinin (Km = 16 microM, kcat = 147 min-1), Met5-Lys6-enkephalin (Km = 375 microM, kcat = 663 min-1), and Leu5-Arg6-enkephalin (Km = 63 microM, kcat = 106 min-1). Although the enzyme shares some properties with other carboxypeptidase B-like enzymes, it is structurally, catalytically, and immunologically distinct from pancreatic carboxypeptidase A or B, human plasma carboxypeptidase N, and carboxypeptidase H ("enkephalin convertase"). To denote that the enzyme is membrane-bound, and to distinguish it from other known carboxypeptidases, we propose the name "carboxypeptidase M." Because of its localization on the plasma membrane and optimal activity at neutral pH, carboxypeptidase M could inactivate or modulate the activity of peptide hormones either before or after their interaction with plasma membrane receptors.     

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.     

The mechanism of the quinone reductase reaction of pig heart lipoamide dehydrogenase.

The relationship between the NADH:lipoamide reductase and NADH:quinone reductase reactions of pig heart lipoamide dehydrogenase (EC 1.6.4.3) was investigated. At pH 7.0 the catalytic constant of the quinone reductase reaction (kcat.) is 70 s-1 and the rate constant of the active-centre reduction by NADH (kcat./Km) is 9.2 x 10(5) M-1.s-1. These constants are almost an order lower than those for the lipoamide reductase reaction. The maximal quinone reductase activity is observed at pH 6.0-5.5. The use of [4(S)-2H]NADH as substrate decreases kcat./Km for the lipoamide reductase reaction and both kcat. and kcat./Km for the quinone reductase reaction. The kcat./Km values for quinones in this case are decreased 1.85-3.0-fold. NAD+ is a more effective inhibitor in the quinone reductase reaction than in the lipoamide reductase reaction. The pattern of inhibition reflects the shift of the reaction equilibrium. Various forms of the four-electron-reduced enzyme are believed to reduce quinones. Simple and 'hybrid ping-pong' mechanisms of this reaction are discussed. The logarithms of kcat./Km for quinones are hyperbolically dependent on their single-electron reduction potentials (E1(7]. A three-step mechanism for a mixed one-electron and two-electron reduction of quinones by lipoamide dehydrogenase is proposed.     

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.     

Purification and properties of biliverdin reductases from pig spleen and rat liver.

Biliverdin reductase was purified from pig spleen soluble fraction to a purity of more than 90% as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme was a monomer protein with a molecular weight of about 34,000. Its isoelectric point was at 6.1-6.2. The enzyme was strictly specific to biliverdin and no other oxiodoreductase activities could be detected in the purified enzyme preparation. The purified enzyme could utilize both NADPH and NADH as electron donors for the reduction of biliverdin. However, there were considerable differences in the kinetic properties of the NADPH-dependent and the NADH-dependent biliverdin reductase activities: Km for NADPH was below 5 microM while that for NADH was 1.5-2 mM; the pH optimum of the reaction with NADPH was 8.5 whereas that of the reaction with NADH was 6.9; Km for biliverdin in the NADPH system was 0.3 microM whereas that in the NADH system was 1-2 microM. In addition, both the NADPH-dependent and NADH-dependent activities were inhibited by excess biliverdin, but this inhibition was far more pronounced in the NADPH system than in the NADH system. IX alpha-biliverdin was the most effective substrate among the four biliverdin isomers, and the dimethylester of IX alpha-biliverdin could not serve as a substrate. Biliverdin reductase was also purified about 300-fold from rat liver soluble fraction. The hepatic enzyme was also a monomer protein with a molecular weight of 34,000 and showed properties quite similar to those of the splenic enzyme as regards the biliverdin reductase reaction. The isoelectric point of the hepatic enzyme, however, was about 5.4. It was assumed that NADPH rather than NADH is the physiological electron donor in the intracellular reduction of IX alpha-biliverdin. The stimulatory effects of bovine and human serum albumins on the biliverdin reductase reactions were also examined.     

Purification and characterization of maleylacetate reductase from Nocardioides simplex 3E utilizing phenoxyalcanoic herbicides 2,4-D and 2,4,5-T.

Maleylacetate reductase was isolated and purified from the Gram-positive strain Nocardioides simplex 3E which is able to utilize the phenoxyalcanoic herbicides 2,4-D and 2,4,5-T. Cells were grown on 2,4-D as the sole carbon source. The enzyme was purified by 380-fold with 3.0% yield. The purified maleylacetate reductase is a homodimer with subunit molecular mass of 37 kD. The enzyme required NADH as a cofactor; the Km for maleylacetate is 25 microM; Vmax (with NADH as cofactor) and kcat are 185 U/mg and 6845 min-1, respectively. The enzyme is very unstable; its pH and temperature optima are at 7.0-7.1 and 50 degrees C, respectively.     

Characterization of the role of lysine 110 of NADH-cytochrome b5 reductase in the binding and oxidation of NADH by site-directed mutagenesis.

An expression vector for bovine NADH-cytochrome b5 reductase was used for site-directed mutagenesis of lysine 110, the residue previously implicated in NADH interactions with this flavoprotein. Replacement of this basic residue with an uncharged glutamine resulted in an increase of 3 orders of magnitude in the Km for NADH and a decrease in kcat of an order of magnitude, strongly implicating lysine 110 in both binding of NADH to the reductase and the orientation of the reduced nicotinamide group for rapid hydride ion transfer to the flavin. Substitution of lysine 110 by histidine, to provide a pH-sensitive positive charge at this position in the neutral pH range, exhibited only a moderate 25-fold increase in Km and a normal kcat at pH 6.0, whereas at pH 8.5 the Km for NADH rose to 238 microM with a decrease of 45% over unmodified enzyme in the kcat. A similar pH sensitivity in the inhibition constant for adenosine diphosphate ribose, lacking only the nicotinamide moiety of NADH, emphasizes the crucial role of the positive charge at this locus and is consistent with charge-pairing of lysine 110 with the pyrophosphate group of NADH or adenosine diphosphate ribose.