Characterization of mutant glucose dehydrogenases with increasing stability.
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Amino Acid Sequence↗
Biomedical subjects
I Urabe
Publications and source records attributed to I Urabe.
Enzymatic properties of isozymes and variants of glucose dehydrogenase from Bacillus megaterium.
Three glucose dehydrogenases (GlcDH) from Bacillus megaterium, GlcDH-I, GlcDH-II and GlcDH-IWG3, were purified from Escherichia coli cells harboring one of the hybrid plasmids, pGDK1, pGDK2 and pGDA3, respectively, pGDK1 and pGDK2 contain two isozyme genes, gdhI and gdhII, respectively, from B. megaterium IAM 1030 and pGDA3 contains an isozyme gene from B. megaterium IWG3; GlcDH-IWG3 is a variant of GlcDH-I. GlcDH-I and GlcDH-II have similar pH/activity profiles and the profile for GlcDH-IWG3 is identical to that of GlcDH-I. The pH/stability profiles of these enzymes show that GlcDH-IWG3 is the most stable enzyme in the acidic region, while GlcDH-II is the most stable in the alkaline region, and GlcDH-I is the most unstable throughout the entire pH range examined. As for thermostability, GlcDH-II is the most resistant against heat inactivation at pH 6.5. The values of the first-order rate constant for heat inactivation at 50 degrees C are 0.27 min-1, 0.05 min-1 and 0.11 min-1 for GlcDH-I, GlcDH-II and GlcDH-IWG3, respectively. Kinetic studies show that these enzymes have similar kinetic constant values except that there are some differences in Kia for NAD(P) and Ka (the limiting Michaelis constant) for NAD; the values of the ratio of Kia for NAD and NADP are 11,340 and 8.7 for GlcDH-I, GlcDH-II and GlcDH-IWG3, respectively. GlcDH-I and GlcDH-IWG3 have very similar substrate specificities and GlcDH-II has a slightly higher specificity for D-glucose and 2-deoxy-D-glucose than the others. The results are discussed on the basis of the amino acid substitutions between the enzymes.
Bacillus megaterium↗
Stability-increasing mutants of glucose dehydrogenase.
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Bacillus megaterium↗
Enzymatic method for measuring the absolute value of oxygen concentration.
An enzymatic method for measuring the absolute concentration of oxygen in aqueous solutions, using 4-hydroxybenzoate 3-monooxygenase and glucose oxidase, is described. The monooxygenase is used for quantitative oxidation of 4-hydroxybenzoate and NADPH with oxygen into 3,4-dihydroxybenzoate and NADP+; the amount of oxygen can be measured as the amount of NADPH decreased by the reaction. The monooxygenase reaction is performed in a syringe, a closed system. After the completion of the monooxygenase reaction, glucose oxidase is added to the assay solution to consume the oxygen from the atmosphere; this enables us to measure the NADPH concentration in the solution spectrophotometrically in an open system and to check the anaerobicity of closed systems. The oxygen concentrations at 25 degrees C of oxygen-saturated and air-saturated water were 1.10 and 0.23 mM, respectively. The value for argon-bubbled water was zero within the experimental error; this justifies the assay system. Thus, it is shown that a sample containing 8 microM-1.1 mM oxygen can be measured by this method.
Aspergillus↗
Stability-increasing mutants of glucose dehydrogenase from Bacillus megaterium IWG3.
A glucose dehydrogenase gene was isolated from Bacillus megaterium IWG3, and its nucleotide sequence was identified. The amino acid sequence of the enzyme deduced from the nucleotide sequence is very similar to the protein sequence of the enzyme from B. megaterium M1286 reported by Jany et al. (Jany, K.-D., Ulmer, W., Froschle, M., and Pfleiderer, G. (1984) FEBS Lett. 165, 6-10). The isolated gene was mutagenized with hydrazine, formic acid, or sodium nitrite, and 12 clones (H35, H39, F18, F20, F191, F192, N1, N13, N14, N28, N71, and N72) containing mutant genes for thermostable glucose dehydrogenase were obtained. The nucleotide sequences of the 12 genes show that they include 8 kinds of mutants having the following amino acid substitutions: H35 and H39, Glu-96 to Gly; F18 and F191, Glu-96 to Ala; F20, Gln-252 to Leu; F192, Gln-252 to Leu and Ala-258 to Gly; N1, Glu-96 to Lys and Val-183 to Ile; N13 and N14, Glu-96 to Lys, Val-112 to Ala, Glu-133 to Lys, and Tyr-217 to His; N28, Glu-96 to Lys, Asp-108 to Asn, Pro-194 to Gln, and Glu-210 to Lys; and N71 and N72, Tyr-253 to Cys. These mutant enzymes have higher stability at 60 degrees C than the wild-type enzyme. The results of this study indicate that the tetrameric structure of glucose dehydrogenase is stabilized by several kinds of mutation, and at least one of the following amino acid substitutions stabilizes the enzyme: Glu-96 to Gly, Glu-96 to Ala, Gln-252 to Leu, and Tyr-253 to Cys.
Amino Acid Sequence↗
Structure and properties of malic enzyme from Bacillus stearothermophilus.
The malic enzyme (EC 1.1.1.38) gene of Bacillus stearothermophilus was cloned in Escherichia coli, and the enzyme was purified to homogeneity from the E. coli clone. In addition to the NAD(P)-dependent oxidative decarboxylation of L-malate, the enzyme catalyzes the decarboxylation of oxalacetate. The enzyme is a tetramer of Mr 200,000 consisting of four identical subunits of Mr 50,000. The pH optima for malate oxidation and pyruvate reduction are 8.0 and 6.0, respectively; and the optimum temperature is 55 degrees C. The enzyme strictly requires divalent metal cations for its activity, and the activity is enhanced 5-7 times by NH4+ and K+. Kinetic study shows that the values of the dissociation constant of the enzyme-coenzyme complex are 77 microM for NAD and 1.0 mM for NADP, indicating that the enzyme has a higher affinity for NAD than for NADP. The nucleotide sequence of the gene and its flanking regions was also found. A single open reading frame of 1434 base pairs encoding 478 amino acids was concluded to be that for the malic enzyme gene because the amino acid composition of the enzyme and the sequence of 16 amino acids from the amino terminus of the enzyme agreed well with those deduced from this open reading frame.
Amino Acid Sequence↗
Synthesis and characterization of 1-substituted 5-alkylphenazine derivatives carrying functional groups.
The following 1-substituted derivatives of 5-methylphenazine and 5-ethylphenazine were synthesized: 1-(3-carboxypropyloxy)-5-methylphenazine (1B), 1-(3-carboxypropyloxy)-5-ethylphenazine (2B), 1-(3-ethoxycarbonylpropyloxy)-5-ethylphenazine (2C) and 1-[N-(2-aminoethyl)carbamoylpropyloxy]-5-ethylphenazine (2D); their spectra, stability and reactivity as electron mediators were investigated, together with those of 5-methylphenazine (1A) and 5-ethylphenazine (2A). The 1-substituted derivatives are all insensitive to light and the derivatives of 5-ethylphenazine are more stable than those of 5-methylphenazine under neutral and alkaline conditions; 2B is the most stable of all the derivatives. The spectral properties of the decomposed compounds showed that photodecomposition of 1A and 2A is associated with hydroxylation at position 1, alkali decomposition of 1A and 1B with elimination of the 5-methyl group and alkali decomposition of 2A, 2B, and 2D with a ring-opening reaction. The second-order rate constant k1 for the reaction of the phenazine derivatives with NADH was measured under steady-state conditions. The k1 values vary depending on the substituents at positions 1 and 5: the values for 1A, 1B, 2A, 2B, 2C and 2D are 1.83 mM-1 s-1, 3.33 mM-1 s-1, 0.75 mM-1 s-1, 1.42 mM-1 s-1, 1.68 mM-1 s-1 and 2.03 mM-1 s-1, respectively. The rate constants, k2 and k3, for the reactions of the reduced form of 2B with oxygen and with 3-(4',5'-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium ion, respectively, were k2 = 1.21 mM-1 s-1 and k3 = 91 mM-1 s-1. These phenazine derivatives have potential applications in the biochemical field.
Alkylation↗
Preparation and kinetic properties of 5-ethylphenazine-poly(ethylene glycol)-NAD+ conjugate, a unique catalyst having an intramolecular reaction step.
5-Ethylphenazine-poly(ethylene glycol)-NAD+ conjugate (EP+-PEG-NAD+) was prepared by linking 1-(3-carboxypropyloxy)-5-ethylphenazine (I) to poly(ethylene glycol)-bound NAD+ (PEG-NAD+) and its kinetic properties were studied. As a reference compound, poly(ethylene glycol)-bound 5-ethylphenazine derivative (III) was also prepared and the effects of poly(ethylene glycol) on the reaction rate of the 5-ethylphenazine moiety with NADH was investigated. The second-order rate constant, k1, of the reaction of III with NADH is 2.78 mM-1 s-1 and is about 1.7 times that of 1-(3-ethoxycarbonylpropyloxy)-5-ethylphenazine (II) with NADH. A similar effect of the attached poly(ethylene glycol) was observed for the reaction of PEG-NADH with I or II. The second-order rate constants, k2 and k3, of the reactions of the reduced form of III with oxygen and with 3-(4',5'-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium ion, respectively, were k2 = 1.22 mM-1 s-1 and k3 = 32 mM-1 s-1; the k2 value is not changed but the k3 value is decreased by the attachment of the polymer. EP+-PEG-NAD+ works as a unique catalyst having an intramolecular reaction step within its turnover cycle in a coupled multi-step reaction system containing malate dehydrogenase, malate, EP+-PEG-NAD+, a tetrazolium salt and oxygen. The first-order rate constant, k4, of the intramolecular reaction was 1.1 s-1. The effects of the covalent linking of the 5-ethylphenazine and the NAD+ moieties were estimated by comparing the value of k4 with that of k1 for the reaction of III with NADH; the effective concentration of the NADH moiety for the 5-ethylphenazine moiety on the same EP+-PEG-NADH molecule (or vice versa) was calculated to be 0.40 mM from the ratio of k4/k1. The values of the rate constants in the coupled multi-step reaction system enable us to understand the dynamic features of the system and the characteristics of EP+-PEG-NAD+ as a catalyst are discussed.
Catalysis↗
Polymerizable NAD derivative and model enzyme reactor with recycling of polyethylene glycol-bound NAD.
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Alcohol Dehydrogenase↗
Anchimeric assistance in the intramolecular reaction of glucose-dehydrogenase-polyethylene glycol NAD conjugate.
Polyethylene glycol-bound derivatives of NAD(P) (PEG-NAD(P)) are water-soluble macromolecular coenzymes used in continuous enzyme reactors. These NAD(P) derivatives have good coenzyme activity for many dehydrogenases, but some enzymes such as glucose dehydrogenase (EC 1.1.1.47) show very low activity with these derivatives (less than 0.1% of that for native NAD(P)). In this work, we prepared a covalently linked glucose-dehydrogenase-polyethylene glycol-NAD conjugate (GlcDH-PEG-NAD) and found that the conjugate shows a much higher reaction rate than that of the native enzyme plus PEG-NAD: the ratio of the reaction rates of GlcDH-PEG-NAD and the native enzyme plus PEG-NAD is calculated to be 10,000-fold at the concentrations of the enzyme subunit and NAD moiety of 0.31 and 0.65 microM, respectively; the rate of the conjugate is even higher than that of the native enzyme plus native NAD. This rate acceleration is due to the increase in the effective concentration of NAD moiety ("anchimeric assistance") and demonstrates the potential of covalent linking for improving the interaction between an enzyme and a coenzyme derivative.
Bacillus megaterium↗
Covalent linking of poly(ethyleneglycol)-bound NAD with Thermus thermophilus malate dehydrogenase. NAD(H)-regeneration unit for a coupled second-enzyme reaction.
Poly(ethyleneglycol)-bound NAD (PEG-NAD) was covalently linked to Thermus thermophilus malate dehydrogenase with a bifunctional reagent, 3,3'-(1,6-dioxo-1,6-hexanediyl)bis-2-thiazolidinethione. The covalently linked malate-dehydrogenase--PEG--NAD complex (MDH-PEG-NAD) was purified by DEAE-Sephadex column chromatography to remove unbound PEG-NAD, and fractionated by blue-Sepharose column chromatography into four preparations: MDH-PEG-NAD I, MDH-PEG-NAD II, MDH-PEG-NAD III and MDH-PEG-NAD IV. The average numbers of NAD moieties covalently bound per subunit of MDH-PEG-NAD I, MDH-PEG-NAD II, MDH-PEG-NAD III and MDH-PEG-NAD IV were 1.2, 1.2, 0.8 and 0.5, respectively, and the values were confirmed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. 60-80% bound NAD moiety of these preparations of MDH-PEG-NAD was reduced by the enzyme moiety in the presence of L-malate, and the specific activity of the enzyme moiety of the preparations was more than 80% that of the native enzyme. MDH-PEG-NAD I has the following properties. The Km value for exogenous NAD is three times that of the native enzyme. The coenzyme activity of its NAD moiety is 20-40% that of native NAD for alcohol and lactate dehydrogenases. The complex catalyzes the oxidation of L-malate in the presence of the redox system of 5-ethylphenazinium ethyl sulfate and a tetrazolium salt with a rate constant of 0.11 s-1. The coenzyme moiety of the complex can also be recycled by coupled reactions of the active site of the same complex and alcohol dehydrogenase. These results indicate that MDH-PEG-NAD works as an NAD(H)-regeneration unit for coupled reactions.
Coenzymes↗
Synthesis of poly(ethylene glycol)-bound NADP by selective modification at the 6-amino group of NADP.
The N-1 position of the adenine ring of NADP was selectively alkylated by the reaction of 2',3'-cyclic NADP with 3-propiolactone to yield 2',3'-cyclic 1-(2-carboxyethyl)-NADP (I). Derivative I was converted to a mixture of the isomers of N6-(2-carboxyethyl)-NADP with their phosphate groups at the 2' or 3' position (IIa and IIb) by chemical reduction, alkaline rearrangement and chemical reoxidation. Carbodiimide coupling of the mixture of IIa and IIb to alpha, omega-diaminopoly(ethylene glycol) gave the 2', 3'-cyclic derivative of poly(ethylene glycol)-bound NADP (III), which was enzymically hydrolyzed to yield poly(ethylene glycol)-bound NADP (PEG-NADP). PEG-NADP has good cofactor activity (16-100% of that of NADP) for NADP-specific and NAD(P)-specific dehydrogenases except isocitrate and glucose dehydrogenases. For NAD-specific enzymes, PEG-NADP has higher cofactor activity than NADP: for horse liver alcohol dehydrogenase, the cofactor activity of PEG-NADP is 40 times that of NADP and 14% of that of NAD. Kinetic studies show that for most of enzymes tested, Km values for PEG-NADP are larger than those for NADP and V values for PEG-NADP are similar to those for NADP. PEG-NADP proved to be applicable in a continuous enzyme reactor, in which reactions of glutamate dehydrogenase and glucose-6-phosphate dehydrogenase were coupled by the recycling of PEG-NADP.
Alkylation↗
Preparation and characterization of NADP derivatives alkylated at 2'-phosphate and 6-amino groups.
Reaction of NADP with 3-propiolactone at pH 6 gave new NADP derivatives carboxyethylated at the 2'-phosphate or 6-amino group, or both: 2'-O-(2-carboxyethyl)phosphono-NAD (I), N6-(2-carboxyethyl)-NADP (II), and 2'-O-(2-carboxyethyl)phosphono-N6-(2-carboxyethyl)-NAD (III). Their structures were assigned on the basis of ultraviolet, 1H-NMR and 31P-NMR spectra, and also treatment with nucleotide pyrophosphatase or alkaline phosphatase. Carbodiimide-promoted reaction of derivative I with 1,2-diaminoethane gave 2'-O-[N-(2-aminoethyl)carbamoylethyl]phosphono-NAD (IV); derivative III gave 2'-O-[N-(2-aminoethyl)carbamoylethyl]phosphono-N6-[N-(2-aminoethyl ) carbamoylethyl]-NAD (IV). The same reaction of derivative II, on the other hand, gave a mixture of N6-[N-(2-aminoethyl)carbamoylethyl]-NADP (Va) and its 3'-phosphate isomer (Vb). The mixture was converted to Va via the 2',3'-cyclic derivative (Vc). Their structures were assigned on the basis of ultraviolet and 1H-NMR spectra, and also treatment with alkaline phosphatase or 3'-nucleotidase. All the NADP derivatives obtained in this work could be reduced with yeast glucose-6-phosphate dehydrogenase.
Alkylating Agents↗
Coenzymic activity of NADP derivatives alkylated at 2'-phosphate and 6-amino groups.
Coenzymic activities of the following NADP derivatives were investigated: 2'-O-(2-carboxyethyl)phosphono-NAD (I), N6-(2-carboxyethyl)-NADP (II), 2'-O-(2-carboxyethyl)phosphono-N6-(2-carboxyethyl)-NAD (III), 2'-O-[N-(2-aminoethyl)carbamoylethyl]phosphono-NAD (IV), N6-[N-(2-aminoethyl)carbamoylethyl]-NADP (Va), 2',3'-cyclic NADP, and 3'-NADP. Derivatives I and IV show the effects of modification at the 2'-phosphate group, and derivatives II and Va show those at the 6-amino group of NADP. As for enzymes, alcohol, isocitrate, 6-phosphogluconate, glucose, glucose-6-phosphate, and glutamate dehydrogenases were used. These enzymes were grouped on the basis of the ratio of the activities for NAD and NADP into NADP-specific enzymes (ratio less than 0.01), NAD(P)-specific enzymes (0.01 less than ratio less than 100), and NAD-specific enzymes (ratio greater than 100). For NADP-specific enzymes, modifications at the 2'-phosphate group of NADP caused great loss of cofactor activity. The relative cofactor activities (NADP = 100%) of derivatives I and IV for these enzymes were 0.5-20 and 0.01-0.5%, respectively. On the other hand, NAD(P)-specific enzymes showed several types of responses to the NADP derivatives. The relative cofactor activities of I and IV for Leuconostoc mesenteroides and Bacillus stearothermophilus glucose-6-phosphate dehydrogenases and beef liver glutamate dehydrogenase were 60-200%; whereas, for B. megaterium glucose dehydrogenase and L. mesenteroides alcohol dehydrogenase, the values were 0.8-8%. For NAD-specific enzymes, these values were 20-50%. The relative cofactor activities of 2',3'-cyclic NADP and 3'-NADP were very low (less than 0.2%) except for beef liver glutamate dehydrogenase, B. stearothermophilus glucose-6-phosphate dehydrogenase, and horse liver alcohol dehydrogenase. Kinetic studies showed that the losses of the cofactor activity of NADP by these modifications were mainly due to the increase of the Km value. The mechanisms of coenzyme specificity of dehydrogenases are discussed. Unlike the 2'-phosphate group, the 6-amino group is common to NAD and NADP, and the effects of modification at the 6-amino group were independent of the coenzyme specificity of enzymes used for the assay. Derivatives II and Va had high relative cofactor activities (65-130%) for most of the enzymes except for isocitrate and glucose dehydrogenases (less than 1%) and L. mesenteroides alcohol dehydrogenase (20-60%). The cofactor activity of derivative III was generally lower than those of I and II.
Alkylation↗
Feasibility study on a continuous environmental gamma-ray monitor using a NaI(Tl) scintillation spectrometer.
Energy spectra of gamma rays due to artificial radionuclides could be estimated by using a NaI(Tl) scintillation spectrometer on the basis of the analysis of natural environmental gamma rays. This analysis system was applied to a continuous gamma-ray monitor in the vicinity of a nuclear facility, and a plume exposure rate due to gaseous effluents of about 6 X 10(-2) Ci hr-1 (2.2 X 10(9) Bq hr-1) could be estimated successfully downwind from the stack.
Air Pollution, Radioactive↗
Steady-state kinetics of coupled two-enzyme reactor with recycling of poly(ethylene glycol)-bound NAD.
The kinetic properties of a continuous enzyme reactor containing rabbit muscle lactate dehydrogenase, horse liver alcohol dehydrogenase and poly(ethylene glycol)-bound NAD (PEG-NAD) were investigated experimentally and theoretically. The enzymes and PEG-NAD were retained in the reactor with an ultrafiltration membrane, and the substrates (pyruvate and ethanol) were fed continuously. The reactions of the dehydrogenases were coupled by the recycling of the cofactor. The steady-state concentration of L-lactate, one of the products, was measured under different experimental conditions and compared with the corresponding theoretical value. The theoretical value was calculated based on a simplified ordered bi-bi mechanism for the individual enzyme reactions, of which kinetic constants were determined by independent kinetic studies. Differences were found between the kinetic constants of the enzymes for NAD(H) and PEG-NAD(H). The steady-state values obtained by continuous operation were lower than those calculated, possibly due to the simplification made for the kinetic model; but there was general agreement between them in the dependence on the experimental conditions. The steady-state behavior of the enzyme reactor was explained semi-quantitatively by the simple kinetic model.
Alcohol Dehydrogenase↗
Inhibition of lactate dehydrogenase activity by polymeric NAD derivatives with different NAD densities.
Polymeric NAD derivatives with different NAD densities were prepared by the copolymerization of NAD N6-[N-(N-acryloyl-1-methoxycarbonyl-5-aminopentyl)-propionamide] with acrylamide in different molar ratios, from 4 x 10(-4) to 1 x 10(-2). The cofactor activities of the polymeric NAD derivatives for lactate, yeast alcohol and horse liver alcohol dehydrogenases decreased with the increase in the NAD density of the polymer, and the lactate dehydrogenase showed no activity for the polymeric NAD derivatives with NAD densities of 2 x 10(-3) and 1 x 10(-2) mol/mol acrylamide polymerized. The polymeric NAD derivatives inhibited the activities of lactate and yeast alcohol dehydrogenases but did not inhibit those of horse liver alcohol and malate dehydrogenases. The polymeric NAD derivatives were competitive inhibitors with respect to NAD for the reaction of lactate dehydrogenase: the inhibition constant decreased from 2.2 microM to 0.02 microM with the increase in the NAD density from 4 x 10(-4) to 1 x 10(-2) mol/mol acrylamide polymerized.
Acrylic Resins↗
Macromolecular derivatives of NAD and their application.
The effects of the properties of polymer chain and NAD density on the cofactor activity of polymeric NAD derivatives were investigated. NAD-N6- [N-(N-acryloyl-l-methoxycarbonyl-5-aminopentyl)-propioamide] (monomeric NAD derivative) was copolymerized with different kinds of vinyl monomers. The resulting polymeric NAD derivatives, except for that prepared with methacrylamide, had similar cofactor activities. These results show that the size and charge of the vinyl monomers do not have a great effect on the cofactor activity of polymeric NAD derivatives. The polymeric NAD derivative prepared by copolymerization with methacrylamide had the highest cofactor activities, and the activities for lactate and yeast alcohol dehydrogenases decreased with an increase in the NAD density of the polymer. These NAD density effects were mainly due to the inhibition by the polymeric NAD derivative with a high NAD density.
Alcohol Oxidoreductases↗