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The crystal structure of glucose dehydrogenase (GlcDH) from Bacillus megaterium IWG3 has been determined to an R-factor of 17.9% at 1.7 Ä resolution. The enzyme consists of four identical subunits, which are similar to those of other short-chain reduc-tases/dehydrogenases (SDRs) in their overall folding and subunit architecture, although cofactor binding sites and subunit interactions differ. Whereas a pair of basic residues is well conserved among NADP+-preferring SDRs, only Arg39 was found around the ade-nine ribose moiety of GlcDH. This suggests that one basic amino acid is enough to determine the coenzyme specificity. The four subunits are interrelated by three mutually perpendicular diad axes (P, Q, and R). While subunit interactions through the P-axis for GlcDH are not so different from those of the other SDRs, those through the Q-axis differ significantly. GlcDH was found to have weaker hydrophobic interactions in the Q-inter-face. Moreover, GlcDH lacks the salt bridge that stabilizes the subunit interaction in the Q-interface in the other SDRs. Hydrogen bonds between Q-axis related subunits are also less common than in the other SDRs. The GlcDH tetramer dissociates into inactive monomers at pH 9.0, which can be attributed mainly to the weakness of the Q-axis interface. [ABSTRACT FROM AUTHOR]

Combinations of substrate-binding sites and catalytic groups constitute various kinds of enzymelike catalysts. The design of such catalysts can be evaluated by the enhancement of the overall catalytic activity by combining these parts into one catalyst. For a catalyst having one substrate-binding site and one catalytic group, an equation was obtained which shows the relationship between the rateacceleration due to the combination, the affinity of the site (1/Kd), intrinsic effective concentration (kin/kcx) and substrate concentration ([S]). The intrinsic effective concentration is the ratio of the firstorder rate constant (kin) of the intramolecular reaction between the catalytic group and the bound substrate and the second-order rate constant (kcx) of the intermolecular reaction between the catalytic group and the free substrate; the value depends on the method of linking the catalytic group and the binding site. This equation provides the following principles for designing catalysts of this type with a considerable grade of rate-acceleration: [S] ≤ kin/kex and (1/10)[S] ≤ Kd ≤ kin/kcx. To increase kin/kcx, the structure of the binding site is required not to reduce the reactivity of the bound substrate, and the linker connecting the binding site and the catalytic group is required to be flexible and to have an appropriate length. A subunit structure is also found to be effective to improve the catalytic activity: the activity of an n-met is at most n² times as high as that of the monomer. As for the substrate-binding sites, the sites of natural enzymes and antibodies are good candidates because various kinds of binding sites with high affinity and specificity to the corresponding substrates are available. In addition, the equation relating the rate-acceleration with Kd, kin/kex, and [S] is used for explaining the catalytic efficiency of enzymes energetically. The principle for designing a multifunctional catalyst having several kinds of binding sites for its substrates and intermediates and several kinds of catalytic groups was then investigated. In this case, the diffusion of the intermediates strongly affects the activity of the multifunctional catalyst, and such a diffusion process was also analyzed. On the basis of these analyses, the following principles were obtained. For a multistep reaction by which a substrate is converted to intermediates successively and finally to a product (linear coupling), the ratio of the dissociation rate constant and the intramolecular rate constant (including the transfer step to the next site) for each intermediate should be made as small as possible; but in practice, it seems very difficult. For a multistep reaction in which reaction steps are coupled by the recycling of the intermediates (cyclic coupling), covalent linking of the intermediates to the catalyst is the best way, and the number of the linked intermediates should be more than that of the binding sites. [ABSTRACT FROM AUTHOR]

5-Ethylphenazine-lactate-dehydrogenase-NAD+ conjugate (EP+-LDH-NAD+) was prepared by linking poly(ethylene glycol)-bound 5-ethylphenazine and poly(ethylene glycol)-bound NAD + to lactate dehydrogenase. The average number of the ethylphenazine moieties bound per molecule of enzyme subunit was 0.46, and that of the NAD+ moieties was 0.32. This conjugate is a semisynthetic enzyme having lactate oxidase activity using oxygen or 3-(4,5-dimethyl-2-thiazolyl)-2,5-dipheny1-2Htetrazolium bromide (MTT) as an electron acceptor; to make such conjugates seems to be a general method for artificially converting a dehydrogenase into an oxidase. When the concentration of oxygen or MTT is varied, the oxidase activity fits the Michaelis-Menten equation with the following kinetic constants: for the reaction system with oxygen, the turnover number per subunit is 2.3 min-1 and Km for oxygen is 1.91 mM; and for the system with MTT, the turnover number is 0.25 min-1 and Km for MTT is 0.076 mM. At the initial steady state of the oxidase reaction, only 2.1% of the NAD+ moieties of the conjugate are in the free state (i.e. not bound in the coenzyme-binding site of the lactate dehydrogenase moiety) and the rest are hidden in the coenzyme site; almost all the NAD+ moieties are in the reduced state. The apparent intramolecular rate constant for the reaction between a free NADH moiety and an oxidized ethylphenazine moiety is 2.3 s-1 and 2.1 s-1 for the systems with oxygen and with MTT, respectively. The apparent effective concentration of the free NADH moiety for the ethylphenazine moiety is 5.5 µM and is much smaller than that (0.34 mM) of the ethylphenazine moiety for the free NADH moiety; this difference is due to the effect of hiding the NADH moiety in the binding site, as the hidden NADH moiety cannot react with the ethylphenazine moiety. [ABSTRACT FROM AUTHOR]

5-Ethylphenazine-poly(ethylene glycol)-glutamate dehydrogenase conjugate (EP+-PEG-GluDH) was prepared by linking poly(ethylene glycol)-bound 5-ethylphenazine to glutamate dehydrogenase. The average number of the ethylphenazine moieties bound/enzyme subunit was 0.7. This conjugate is a semisynthetic enzyme having NADH oxidase activity; the ethylphenazine moiety works as a catalytic group, and the coenzyme-binding site of glutamate dehydrogenase works as a substrate-binding site. The effects of the presence of the substrate-binding site near the catalytic group were studied by using EP+-PEG-GluDH. Before the preparation of the conjugate, the reactivity of NADH bound in the coenzyme-binding site toward the ethylphenazine moiety was estimated for glutamate and lactate dehydrogenases. The results show that the NADH molecule bound in the site of glutamate dehydrogenase reacts with EP+-PEG at a rate of 43% of that of free NADH, but the NADH molecule bound in lactate dehydrogenase does not react with 1-(3-carboxypropyloxy)-5-ethylphenazine. Therefore, glutamate dehydrogenase was used as the substrate-binding site of the semisynthetic NADH oxidase. The results of the kinetic analysis of the activity of EP+-PEG-GluDH show that the apparent turnover number of the active site is 0.38 s-1, which corresponds to the apparent intramolecular rate constant of the oxidation of NADH bound in the active site. The apparent effective concentration of bound NADH for the catalytic group of the ethylphenazine moiety is 0.33 mM. This means that the presence of the substrate-binding site near the catalytic group increases the local NADH concentration by at most 0.33 mM, and this is the rate-accelerating effect of the binding site. [ABSTRACT FROM AUTHOR]

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 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 <0.01), NAD(P)-specific enzymes (0.01 < ratio < 100), and NAD and NADP into NADP-specific enzymes (ratio <0.01), NAD(P)-specific enzymes (0.01 < ratio < 100), and NAD-specific enzymes (ratio > 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 0.1-0.5%, respectively. On the other hand, NAD(P)-specificenzymes 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 dehydrogenase 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. [ABSTRACT FROM AUTHOR]

The kinetic properties of a continuous enzyme reactor containing rabbit muscle lactate dehydrogenasc, horse liver alcohol dehydrogenase and poly(ethylene glycol)abound NAD (PEG-NAD) were investigated experimentally and theoretically. The enzymes and PEG-NA!) 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 NAID(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. [ABSTRACT FROM AUTHOR]

The nucleotide sequence of the β-xylosidase (xynB) gene from Bacillus pumilus has been reported previously [Moriyama, H., Fukusaki, E., Crespo, J. C., Shinmyo, A. & Okada, H. (1987) Eur. J. Biochem. 166, 539–545]. However, the sequence identified in the present study is quite different from the previously reported one. The total length of the PstI-EcoBI fragment of a plasmid pOXN295 containing the xynB gene is 2201 bp from our sequencing, while the length of the fragment in the previous data was 2466 bp. The sequences are similar in the N-terminal (500 bp) and C-terminal (260 bp) regions, but those in the central region are completely different. From the following observations, the previous sequence seems to have no reliable experimental basis. First, the restriction sites observed for pOXN295 are quite different from the sites deduced from the sequence. Second, the amino acid composition deduced from the sequence and the composition identified by amino acid analysis of the purified β-xylosidase are very different. It is confirmed, on the other hand, that our new sequence agrees well with these experimental data. The enzyme was purified to homogeneity from Bacillus pumilus and Escherichia coli harboring a hybrid plasmid which highly expresses the xynB gene. The molecular mass of the enzyme was estimated to be 190 kDa by high performance gel filtration chromatography using TSK-G3000SW and 56 kDa by SDS/polyacrylamide gel electrophoresis. The pH optimum was 7.0, and the optimum temperature was 40 °C. The Vm value was estimated to be 1.23±0.14 μkat/mg (for p-nitrophenyl β-D-xyloside) and 0.14±0.011 μkat/mg (for xylobiose), while Km was estimated to be 3.9±0.59 mM (for p-nitrophenyl β-D-xyloside) and 8.9±1.19 mM (for xylobiose). [ABSTRACT FROM AUTHOR]

The structural genes of two homologous enzymes, 6-aminohexanoate-dimer hydrolase (EII; nylB) and its evolutionally related protein EII' (nylB') of Flavobacterium sp. KI72 have an open reading frame encoding a peptide of 392 amino acids, of which 47 are different, and conserved restriction sites. The specific activity of EII towards 6-aminohexanoate dimer is about 1000-fold that of EII'. Construction of various hybrid genes obtained by exchanging fragments flanked by conserved restriction sites of the two genes demonstrated that two amino acid replacements in the EII' enzyme, i.e. Gly181 → Asp (EII type) and His266 → Asn (EII type), enhanced the activity toward 6-aminohexanoate dimer 1000-fold. [ABSTRACT FROM AUTHOR]

Reports on the covalent linking of poly(ethyleneglycol)-bound NAD with Thermus thermophilus malate dehydrogenase. Confirmation by sodium dodecyl sulfate/polyacrylamide gel electrophoresis; Specific activity of the enzyme moiety of the preparations; Coenzyme activity of its NAD moiety.

An experimental evolution with selection based on binding affinity to DNA was carried out on a library of phage-displayed random polypeptides of about 140 amino acid residues. First, we constructed a system to artificially evolve phage-displayed random polypeptides toward binding to a target DNA containing a restriction enzyme site, in which random polypeptides capable of binding the DNA were recovered as complexes with the target DNA by digestion with the restriction enzyme. The experimental evolution cycle, including the above selection system and random mutagenesis for generating the next mutant library, was repeated until the fourth generation. The ability to bind to the DNA was enhanced per generation. In the fourth generation, convergence of the selected clones to a dominant sequence was observed. These results indicate that the newly constructed selection system is effective for exploring the evolvability of random polypeptides towards DNA-binding proteins. [Copyright &y& Elsevier]