Identity of dimeric dihydrodiol dehydrogenase as NADP+-dependent d-xylose dehydrogenase in pig liver

Dimeric dihydrodiol dehydrogenases (DDs, EC 1.3.1.20), which oxidize trans-dihydrodiols of aromatic hydrocarbons to the corresponding catechols, have been molecularly cloned from human intestine, monkey kidney, pig liver, dog liver, and rabbit lens. A comparison of the sequences with the DNA sequences in databases suggested that dimeric DDs constitute a novel protein family with 20 gene products. In addition, it was found that dimeric DD oxidizes several pentoses and hexoses, and the specificity resembles that of NADP⁺-dependent d-xylose dehydrogenase (EC 1.1.1.179) of pig liver. The inhibition of d-xylose dehydrogenase activity in the extracts of monkey kidney, dog liver and pig liver, its co-purification with dimeric DD activity from pig liver, and kinetic analysis of the d-xylose reduction by pig dimeric DD indicated that the two enzymes are the same protein.     

Kinetic analysis of enzymatic hydrolysis of crystalline cellulose by cellobiohydrolase using an amperometric biosensor

An amperometric biosensor for the detection of cellobiose has been introduced to study the kinetics of enzymatic hydrolysis of crystalline cellulose by cellobiohydrolase. By use of a sensor in which pyrroloquinoline quinone-dependent glucose dehydrogenase was immobilized on the surface of electrode, direct and continuous observation of the hydrolysis can be achieved even in a thick cellulose suspension. The steady-state rate of the hydrolysis increased with increasing concentrations of the enzyme to approach a saturation value and was proportional to the amount of the substrate. The experimental results can be explained well by the rate equations derived from a three-step mechanism consisting of the adsorption of the free enzyme onto the surface of the substrate, the reaction of the adsorbed enzyme with the substrate, and the liberation of the product. The catalytic constant of the adsorbed enzyme was determined to be 0.044+/-0.011s(-1).     

Role of catalytic residues in enzymatic mechanisms of homologous ketosteroid isomerases

Ketosteroid isomerase (KSI) is one of the most proficient enzymes catalyzing an allylic isomerization reaction at a diffusion-controlled rate. In this study of KSI, we have detailed the structures of its active site, the role of various catalytic residues, and have explained the origin of the its fast reactivity by carrying out a detailed investigation of the enzymatic reaction mechanism. This investigation included the X-ray determination of 15 crystal structures of two homologous enzymes in free and complexed states (with inhibitors) and extensive ab initio calculations of the interactions between the active sites and the reaction intermediates. The catalytic residues, through short strong hydrogen bonds, play the role of charge buffer to stabilize the negative charge built up on the intermediates in the course of the reaction. The hydrogen bond distances in the intermediate analogues are found to be about 0.2 A shorter in the product analogues both experimentally and theoretically.     

Chorismate mutase-prephenate dehydrogenase from Escherichia coli. 1. Kinetic characterization of the dehydrogenase reaction by use of alternative substrates

The bifunctional enzyme involved in tyrosine biosynthesis, chorismate mutase-prephenate dehydrogenase, has been isolated from extracts of a plasmid-containing strain of Escherichia coli K12 and purified to homogeneity by a modified procedure that involves chromatography on both Matrex Blue A and Sepharose-AMP. Detailed studies of the dehydrogenase reaction have been undertaken with analogues of prephenate that act as substrates. The analogues, which included two of the four possible diastereoisomers of 1-carboxy-4-hydroxy-2-cyclohexene-1-propanoate (deoxodihydroprephenate) as well as D- and L-arogenate, were synthesized chemically. As judged by their V/K values, all analogues were poorer substrates than prephenate. The order of their effectiveness as substrates is prephenate greater than one isomer of 1-carboxy-4-hydroxy-2-cyclohexene-1-propanoate greater than L-arogenate greater than other isomer of 1-carboxy-4-hydroxy-2-cyclohexene-1-propanoate greater than D-arogenate. Thus the dehydrogenase activity is dependent on the degree and position of unsaturation in the ring structure of prephenate as well as on the type of substitution on the pyruvyl side chain. With prephenate as a substrate, the reaction is irreversible because it involves oxidative decarboxylation. By contrast, 1-carboxy-4-hydroxy-2-cyclohexene-1-propanoate undergoes only a simple oxidation, and thus, with this substrate, the reaction is reversible. Steady-state velocity data, obtained by varying substrates over a range of higher concentrations, suggest that the dehydrogenase reaction conforms to a rapid equilibrium, random mechanism with 1-carboxy-4-hydroxy-2-cyclohexene-1-propanoate as a substrate in the forward reaction or with the corresponding ketone derivative as a substrate in the reverse direction. The initial velocity patterns obtained by varying prephenate or 1-carboxy-4-hydroxy-2-cyclohexene-1-propanoate over a range of lower concentrations, at different fixed concentrations of NAD, were nonlinear and consistent with a unique model that is described by a velocity equation which is the ratio of quadratic polynomials. An equilibrium constant of 1.4 x 10(-7) M for the reaction in the presence of 1-carboxy-4-hydroxy-2-cyclohexene-1-propanoate indicates that the equilibrium lies very much in favor of ketone production.     

Kinetic characterization of the pyruvate and oxoglutarate dehydrogenase complexes from human heart

The Michaelis constant values for the highly purified pyruvate dehydrogenase complex (PDC) from human heart are 25, 13 and 50 microM for pyruvate, CoA and NAD, respectively. Acetyl-CoA produces a competitive inhibition of PDC (Ki = 35 microM) with respect to CoA, whereas NADH produces the same type of inhibition with respect to NAD (Ki = 36 microM). The oxoglutarate dehydrogenase complex (OGDC) from human heart has active sites with two different affinities for 2-oxoglutarate ([S]0.5 of 30 and 120 microM). ADP (1 mM) decreases the [S]0.5 values by a half. The inhibition of OGDC (Ki = 81 microM) by succinyl-CoA is of a competitive type with respect to CoA (Km = 2.5 microM), whereas that of NADH (Ki = 25 microM) is of a mixed type with respect to NAD (Km = 170 microM).     

Purification and characterization of fungal and mammalian phosphomannose isomerases

Phosphomannose isomerase (PMI) is essential for the production of yeast cell walls. An inhibitor which inhibits the fungal enzyme without altering the activity of the mammalian enzyme would be a potential fungicidal agent, increasingly important in view of the increasing mortality from visceral mycoses in immunosuppressed patients. We have purified human, porcine, andCandida albicans enzymes 29,000-fold to homogeneity, and characterized their physical properties, as well as their kinetic parameters, inhibition constants, andpH dependences. Surprisingly, in view of the large differences betweenPseudomonas aerugenosa andSaccharomyces cerevisiae PMI, the human andC. albicans enzymes are almost identical. We suggest therefore that species-selective inhibition of the fungal rather than mammalian enzyme may require molecules which bind away from the substrate binding pocket of the enzyme.Abbreviations PMI phosphomannose isomerase - Tris/HCl tris(hydroxymethyl)aminomethane - Hepes 4-(2-hydroxyethyl)-piperazine-1-ethanesulfonic acid - Mes 2-(N-morphilino) ethanesulfonic acid     

Structural and enzymatic characterization of Bacillus subtilis R,R-2,3-butanediol dehydrogenase

2,3-butanediol dehydrogenase (BDH, EC 1.1.1.76) also known as acetoin reductase (AR, EC 1.1.1.4) is the key enzyme converting acetoin (AC) into 2,3-butanediol (BD) and undertaking the irreversible conversion of diacetyl to acetoin in various microorganisms. The existence of three BDHs (R,R-, meso-, and S,S-BDH) product different BD isomers. Catalyzing mechanisms of meso- and S,S-BDH have been understood with the assistance of their X-ray crystal structures. However, the lack of structural data for R,R-BDH restricts the integral understanding of the catalytic mechanism of BDHs. In this study, we successfully crystallized and solved the X-ray crystal structure of Bacillus subtilis R,R-BDH. A zinc ion was found locating in the catalytic center and coordinated by Cys37, His70 and Glu152, helping to stabilize the chiral substrates observed in the predicted molecular docking model. The interaction patterns of different chiral substrates in the molecular docking model explained the react priority measured by the enzyme activity assay of R,R-BDH. Site-directed mutation experiments determined that the amino acids Cys37, Thr244, Ile268 and Lys340 are important in the catalytically active center. The structural information of R,R-BDH presented in this study accomplished the understanding of BDHs catalytic mechanism and more importantly provides useful guidance for the directional engineering of R,R-BDH to obtain high-purity monochiral BD and AC.     

Enzymatic assays: optimization of systems by using pyruvate kinase and lactate dehydrogenase as auxiliary enzymes

A method to optimize enzymatic assays by using pyruvate kinase and lactate dehydrogenase enzymes is presented and applied to mitochondrial ATPase as an example. Optimum amounts of auxiliary enzymes, to obtain either a 99% of the initial rate in a given time (t99) or a given lag period (L), are calculated from their apparent Michaelis constants (Kapp) in the medium used and their prices per enzymatic international unit.     

Kinetic study on the enzymatic resolution of homophenylalanine ester using ionic liquids

Two ionic liquids (ILs) were investigated as novel media for the enzymatic resolution of amino acid ester to obtain enantiomeric amino acid homophenylalanine. The effects of solvent nature, polarity, and concentration on the kinetic resolution were investigated. With change in solvent concentration, a systematic study shows that an improved enzyme activity can be obtained by adjusting these solvent parameters.     

Kinetic regulation of yeast NAD-specific isocitrate dehydrogenase by citrate

The present results suggest that the enzyme modifier citrate and the substrate isocitrate are bound at different sites on yeast NAD-specific isocitrate dehydrogenase and that citrate diminishes the binding of the positive effector 5'-AMP, thereby causing a decreased rate of enzyme catalysis. This interpretation differs from the earlier proposal that citrate can replace isocitrate at an activator site on the enzyme and can cause inhibition by binding at its catalytic site [Atkinson et al. (1965) J. Biol. Chem. 240, 2682]. The present proposal is supported by the following observations: At constant subsaturating levels of isocitrate, NAD+, and Mg2+ without AMP, up to 10 mM citrate was an activator and not an inhibitor. Citrate decreased velocity for AMP-activated enzyme; however, with increasing citrate the specific activity with AMP asymptotically approached but did not decrease below the level of the enzyme maximally activated by citrate in the absence of AMP. When added singly, AMP decreased S0.5 for isocitrate without changing the Hill number (n), whereas citrate lowered n without changing S0.5 for isocitrate. The difference in action of these modifiers indicated that they were bound at separate sites on the enzyme. The binding of citrate appeared to cause a conformational change in the protein that lowered the enzyme's affinity for AMP. This was consistent with the findings that citrate (or the citrate agonist fluorocitrate) (i) resulted in an increase in S0.5 for isocitrate with the AMP-activated enzyme and (ii) decreased binding of the positive effector analogue TNP-AMP as measured by fluorescence change.(ABSTRACT TRUNCATED AT 250 WORDS)     

Induction of aldose reductase and polyol dehydrogenase activities in Aureobasidium pullulans by d-xylose,l-arabinose and d-galactose

Summary The induction of aldose reductase and polyol dehydrogenase activities by d-xylose, l-arabinose, d-galactose and d-glucose was studied in the yeast-like organism Aureobasidium pullulans CCY 27-1-26. d-xylose and l-arabinose induced two distinct NADPH-dependent aldose reductases and the inducing saccharide was simultaneously the most efficient substrate for the corresponding enzymatic reaction. Polyol dehydrogenase induced by d-xylose, l-arabinose and d-galactose was strictly NAD⁺-dependent and required only xylitol as a substrate of the enzymatic reaction. l-Arabitol did not act as a substrate for l-arabinose-induced polyol dehydrogenase either in the presence of NAD⁺ or NADP⁺.     

Kinetic mechanism of glutaryl-CoA dehydrogenase

Glutaryl-CoA dehydrogenase (GCD) is a homotetrameric enzyme containing one noncovalently bound FAD per monomer that oxidatively decarboxylates glutaryl-CoA to crotonyl-CoA and CO2. GCD belongs to the family of acyl-CoA dehydrogenases that are evolutionarily conserved in their sequence, structure, and function. However, there are differences in the kinetic mechanisms among the different acyl-CoA dehydrogenases. One of the unanswered aspects is that of the rate-determining step in the steady-state turnover of GCD. In the present investigation, the major rate-determining step is identified to be the release of crotonyl-CoA product because the chemical steps and reoxidation of reduced FAD are much faster than the turnover of the wild-type GCD. Other steps are only partially rate-determining. This conclusion is based on the transit times of the individual reactions occurring in the active site of GCD.     

Overproduction of d-xylose isomerase in Escherichia coli by cloning the d-xylose isomerase gene

The Escherichia coli d-xylose isomerase (d-xylose ketol-isomerase, EC 5.3.1.5) gene, xylA, has been cloned on various E. coli plasmids. However, it has been found that high levels of overproduction of the d-xylose isomerase, the protein product of the xylA gene, cannot be accomplished by cloning the intact gene on high copy-number plasmids alone. This is believed to be due to the fact that the expression of the gene through its natural promoter is highly regulated in E. coli. In order to overcome this, the xylA structural gene has been fused with other strong promoters such as tac and lac, resulting in the construction of a number of fused genes. Analysis of the E. coli transformants containing the fused genes, cloned on high copy-number plasmids, indicated that a 20-fold overproduction of the enzyme can now be obtained. It is expected that overproduction of the enzyme in E. coli can still be substantially improved through additional manipulation with recombinant DNA techniques.     

Kinetic studies of formate dehydrogenase

1. The kinetic mechanism of formate dehydrogenase is a sequential pathway. 2. The binding of the substrates proceeds in an obligatory order, NAD(+) binding first, followed by formate. 3. It seems most likely that the interconversion of the central ternary complex is extremely rapid, and that the rate-limiting step is the formation or possible isomerization of the enzyme-coenzyme complexes. 4. The secondary plots of the inhibitions with HCO(3) (-) and NO(3) (-) are non-linear, which suggests that more than one molecule of each species is able to bind to the same enzyme form. 5. The rate of the reverse reaction with carbon dioxide at pH6.0 is 20 times that with bicarbonate at pH8.0, although no product inhibition could be detected with carbon dioxide. The low rate of the reverse reaction precluded any steady-state analysis as the enzyme concentrations needed to obtain a measurable rate are of the same order as the K(m) values for NAD(+) and NADH.     

Analysis of rat cytosolic 9-cis-retinol dehydrogenase activity and enzymatic characterization of rat ADHII

We report the characterization of two enzymes that catalyze NAD(+)-dependent 9-cis-retinol dehydrogenase activity in rat liver cystol. Alcohol dehydrogenase class I (ADHI) contributes > 80% of the NA D+-dependent 9-cis-retinol dehydrogenase activity recovered, whereas alcohol dehydrogenase class II (ADHII), not identified previously at the protein level, nor characterized enzymatically in rat, accounts for approximately 2% of the activity. Rat ADHII exhibits properties different from those described for human ADHII. Moreover, rat ADHII-catalyzed rates of ethanol dehydrogenation are markedly lower than octanol or retinoid dehydrogenation rates. Neither ethanol nor 4-methylpyrazole inhibits the 9-cis-retinol dehydrogenase activity of rat ADHII. We propose that ADHII represents the previously observed additional retinoid oxidation activity of rat liver cytosol which occurred in the presence of either ethanol or 4-methylpyrazole. We also show that human and rat ADHII differ considerably in enzymatic properties.     

Kinetic analysis of enzymatic chiral resolution by progress curve evaluation

The present study deals with kinetic modeling of enzyme-catalyzed reactions by integral progress curve analysis, and shows how to apply this technique to kinetic resolution of enantiomers. It is shown that kinetic parameters for both enantiomers and the enantioselectivity of the enzyme may be obtained from the progress curve measurement of a racemate only.A parameter estimation procedure has been established and it is shown that the covariance matrix of the obtained parameters is a useful statistical tool in the selection and verification of the model structure. Standard deviations calculated from this matrix have shown that progress curve analysis yields parameter values with high accuracies.Potential sources of systematic errors in (multiple) progress curve analysis are addressed in this article. Amongst these, the following needed to be dealt with: (1) the true initial substrate concentrations were obtained from the final amount of product experimentally measured (mass balancing); (2) systematic errors in the initial enzyme concentration were corrected by incorporating this variable in the fitting procedure as an extra parameter per curve; and (3) enzyme inactivation is included in the model and a first-order inactivation constant is determined.Experimental verification was carried out by continuous monitoring of the hydrolysis of ethyl 2-chloropropionate by carboxylesterase NP and the alpha-chymotrypsin-catalyzed hydrolysis of benzoylalanine mathyl ester in a pH-stat system. Kinetic parameter values were obtained with high accuracies and model predictions were in good agreement with independent measurements of enantiomeric excess values or literature data. (c) 1994 John Wiley & Sons, Inc.