Kinetic modeling of omega-transamination for enzymatic kinetic resolution of alpha-methylbenzylamine

A kinetic model for omega-transaminase from Bacillus thuringiensis JS64 was developed by using the King-Altman method to simulate the kinetic resolution of alpha-methylbenzylamine (alpha-MBA). Starting from a ping-pong bi-bi mechanism, a complete kinetic model including substrate inhibition only in the reverse reaction (i.e., transamination between acetophenone and L-alanine) was developed. The asymmetric synthesis of (S)-alpha-MBA proved to be difficult due to a much lower maximum reverse reaction rate than the maximum forward reaction rate, thermodynamically exergonic forward reaction (i.e., transamination between (S)-alpha-MBA and pyruvate), and the severe product and substrate inhibition of the reverse reaction. Experimental values for kinetic parameters show that the product inhibition constant of (S)-alpha-MBA is the most important parameter on determining the resolution reaction rate, suggesting that the resolution reaction rate will be very low unless (S)-alpha-MBA strongly inhibits the reverse reaction. Using the kinetic model, the kinetic resolution of alpha-MBA in aqueous buffer was simulated, and the simulation results showed a high degree of consistency with experimental data over a range of reaction conditions. Various simulation results suggest that the crucial bottleneck in the kinetic resolution of alpha-MBA lies mainly in the accumulation of acetophenone in reaction media as the reaction proceeds, whereas L-alanine exerts a little inhibitory effect on the reaction. The model predicts that removing acetophenone produced during the reaction can enhance the reaction rate dramatically. Indeed, the biphasic reaction system is capable of extracting acetophenone from the aqueous phase, showing a much higher reaction rate compared to a monophasic reaction system. The kinetic model was also useful in predicting the properties of other, better enzymes as well as the optimal concentrations of amino acceptor and enzyme in the resolution reaction.     

Sensitivity of the (Na+ + k+)-atpase to state-dependent inhibitors. Effects of digitonin and Triton X-100

Treatment of a purified (NA+ + 5+)-ATPase preparation from dog kidney with digitonin reduced enzymatic activity, with the (Na+ + k+)-atpase reaction inhibited more than the K+-phosphatase reaction that is also catalyzed by this enzyme. Under the usual assay conditions oligomycin inhibits the (Na+ + k+)-atpase reaction but not the K+-phosphatase reaction; however, treatment with digitonin made the K+-phosphatase reaction almost as sensitive to oligomycin as the (Na+ + k+)-atpase reaction. The non-ionic detergents, Triton X-100, Lubrol WX and Tween 20, also conferred sensitivity to oligomycin on the K+-phosphatase reaction (in the absence of oligomycin all these detergents, unlike digitonin, inhibited the K+-phosphatase reaction more than the (Na+ + k+)-atpase reaction). Both digitonin and Triton markedly increased the K0.5 for K+ as activator of the K+-phosphatase reaction, with little effect on the K0.5 for K+ as activator of the (Na+ + k+)-ATpase reaction. In contrast, increasing the K0.5 for K+ in the K+-phosphatase reaction by treatment of the enxyme with acetic anhydride did not confer sensitivity to oligomycin. Both digitonin and Triton also increased the inhibition of the K+-phosphatase reaction by ATP and increased the inhibition by inorganic phosphate and vanadate. These observations are interpreted as digitonin and Triton favoring the E1 conformational state of the enzyme (manifested by sensitivity to oligomycin and a greater affinity for ATP at the low-affinity substrate sites), as opposed to the E2 state (manifested by insensitivity to oligomycin, greater sensitivity to phosphate and vanadate, and a lower K0.5 for K+ in the K+-phosphatase reaction). In addition, digitonin blocked activation of the phosphatase reaction by Na+ plus CTP. This effect is consistent with digitonin dissociating the catalytic subunits of the enzyme, the interaction of which may be essential for activation by Na+ plus nucleotide.     

Synthesis of a new enantiomerically pure P-chiral phosphine and its use in probing the mechanism of the Mitsunobu reaction

A new enantiomerically pure P-chiral phosphine, (S)-cyclohexylmethyl- (1-naphthyl)phosphine (1) was prepared by phosphine-borane methodology and used in a mechanistic study of the Mitsunobu reaction. Enantiomerically enriched (S)-cyclo- hexylmethyl(1-naphtyl)phosphine oxide (8) is obtained if the reaction proceeds through the phosphonium salt 4, whereas the intermediate dialkoxyphosphorane 5 leads to racemic phosphine oxide 8. The results of the experiments including the variation of the reaction conditions and the natures of alcohol and carboxylic acid used in the Mitsunobu reaction prove the competition of two alternative mechanisms (reaction via 4 or 5) on the second stage of the Mitsunobu reaction.     

Effect of food reductones on the generation of the pyrazine cation radical and on the formation of the mutagens in the reaction of glucose, glycine and creatinine

One of the possible pathways of the formation of mutagens in heated foods is through the pyrazine cation radical generated in the early stage of the Maillard reaction. The aim of the present study was to elucidate how food reductones contribute to the pyrazine cation radical generation in the reaction of glucose (Glc) and glycine (Gly), and to the formation of the mutagens in the reaction of Glc, Gly and creatinine. Electron spin resonance (ESR) studies showed that fragrant reductones, 2,5-dimethyl-4-hydroxy-3(2H)-furanone (DMHF) and 4-hydroxy-2(or 5)-ethyl-5(or 2)-methyl-3(2H)-furanone (HEMF), generated in the Maillard reactions, enhanced the generation of the pyrazine cation radical in the reaction of Glc and Gly, and the reaction of DMHF or HEMF with Gly generated a larger amount of the pyrazine cation radical than the reaction of Glc and Gly, indicating that the furanones were intermediates of the pyrazine cation radical. By contrast, food antioxidants, ascorbic acid and erythorbic acid, effectively scavenged the pyrazine cation radical generated in the reaction of Glc and Gly. DMHF and HEMF were not effective to modulate the mutagen formation in the reaction of Glc, Gly and creatinine, and the mutagenicity produced in the reaction of DMHF or HEMF, Gly and creatinine was lower than that produced in the reaction of Glc, Gly and creatinine. On the other hand, ascorbic acid and erythorbic acid were effective to decrease the mutagen formation in the reaction of Glc, Gly and creatinine.     

Kinetic study on the reaction mechanism of pantothenase: existence of an acyl-enzyme intermediate and role of general acid catalysis.

A kinetic study was performed on the reaction mechanism of pantothenase (EC 3.5.1.22) catalyzed hydrolysis of the pantothenic acid. A nonlinear progress curve is derived if the reaction occurs at low buffer concentrations. The nonlinearity is due to partial reversibility of the reaction; an acylenzyme (pantoyl-enzyme) is formed during the reaction, and beta-alanine, the other end product, is able to react with the acyl-enzyme and return back to pantothenate. The dependence of the beta-alanine return reaction on buffer concentration and on pH suggests a general acid catalysis during the reaction. A reaction mechanism is suggested, in which the -NH3+ form of beta-alanine participates in the return reaction, and the deacylation of the acyl-enzyme is acid catalyzed.     

Kinetics of the reaction of nitroblue tetrazolium reduction by human blood neutrophils

Kinetics of Nitro blue tetrazolium (NBT) reduction to diformasan by neutrophils was investigated using 27 samples of human blood. Analysis of alteration in the share of activated neutrophils (ANP) and activated neutrophil index (ANI) was done in relation to the reaction time. The former reaction is an irreversible reaction of zero (pseudozero) order, while the latter is an irreversible reaction of the first (pseudofirst) order. It has been found out that an induced NBT reduction occurs in parallel with a spontaneous reaction, and that neutrophils have essentially different oxidizing power. The kinetic approach enabled us to discover some indices (NBT quantity involved in the reaction, and reaction speed constant of the first order) which in different samples varied within broader limits than ANP or ANI (within the limits of an order), i.e. provided a possibility to make a more delicate analysis of processes in neutrophils.     

A comparative study on the thermal decomposition of four cereal straws in an oxidizing atmosphere

The thermal decomposition characteristics of four types (wheat, barley, oats and rye) of cereal straws were studied. Two varieties from each type of straw were used. The thermal degradation behaviours and kinetic parameters (order of reaction, activation energy and preexponential factor) of the straws were compared. Two distinct reaction zones were observed for all types and varieties of straws. Thermal degradation rates in the first reaction zone were significantly higher than those in the second reaction zone. The activation energy was in the ranges of 80–102 kJ/mol and 34–75 kJ/mol, whereas the order of reaction was in the ranges of 1·3–2·3 and 0·1–0·7 for the first and second reaction zones, respectively. The Shaw variety of oats straw had the highest activation energies (102 and 75 kJ/mol) and reaction orders (2·3 and 0·7) in both the first and second reaction zones, respectively. The lowest activation energy (80 kJ/mol) and order of reaction (1·3), in the first reaction zone, corresponded to Absolvant and Monopol wheat straws. The activation energies and reaction orders of barley and rye straws were in the ranges of 85–94 kJ/mol and 1·9–2·3, respectively. There was not any significant difference between the rate constants of the straw varieties, in the first reaction zone. However, oats straws had significantly higher rate constants in the second reaction zone as compared to the rate constants of wheat, barley and rye straws.     

Enzymatic peptide synthesis in organic media: a comparative study of water-miscible and water-immiscible solvent systems.

Peptide synthesis was carried out in a variety of organic solvents with low contents of water. The enzyme was deposited on the support material, celite, from an aqueous buffer solution. After evaporation of the water the biocatalyst was suspended in the reaction mixtures. The chymotrypsin-catalyzed reaction between Z-Phe-OMe and Leu-NH2 was used as a model reaction. Under the conditions used ([Z-Phe-OMe]0 less than or equal to 40 mM, [Leu-NH2]0/([Z-Phe-OMe]0 = 1.5) the reaction was first order with respect to Z-Phe-OMe. Tris buffer, pH 7.8, was the best buffer to use in the preparation of the biocatalyst. In water-miscible solvents the reaction rate increased with increasing water content, but the final yield of peptide decreased due to the competing hydrolysis of Z-Phe-OMe. Among the water-miscible solvents, acetonitrile was the most suitable, giving 91% yield with 4% (by vol.) water. In water-immiscible solvents the reaction rate and the product distribution were little affected by water additions in the range between 0% and 2% (vol. %) in excess of water saturation. The reaction rates correlated well with the log P values of the solvent. The highest yield (93%) was obtained in ethyl acetate; in this solvent the reaction was also fast. Under most reaction conditions used the reaction product was stable; secondary hydrolysis of the peptide formed was normally negligible. The method presented is a combination of kinetically controlled peptide synthesis (giving high reaction rates) and thermodynamically controlled peptide synthesis (giving stable reaction products).     

Development of two inbred strains of rats and characteristics of their skin reactions.

Two inbred strains of rat (Donryu and Sprague-Dawley strains) were developed. The skin reactions of these strains immunized with M. tuberculosis, hen egg albumin (OVA) or hen egg lysozyme and challenged with the purified protein derivative (PPD) or each antigen were even and uniform. The Donryu strain showed a typical Arthus reaction with petechiae and edema and a negligible delayed skin reaction, whereas the Sprague-Dawley strain showed a poor Arthus reaction and a typical delayed skin reaction with central necrosis and induration. The Arthus reaction or delayed skin reaction could be passively transferred to recipient rats of each strain by immune sera or sensitized peritoneal exudate cells (PEC), respectively.     

Substrate interactions with the alpha-subunit of the Escherichia coli tryptophan synthase. A study of the activity of mutant alpha-subunits.

Extracts of 19 trpA mutant strains of Escherichia coli were examined for their relative activity in the reversible aldolytic reaction catalyzed by the trpA gene product, the α-subunit of tryptophan synthase, in combination with the β-subunit of this enzyme. The specific activities in this reaction, indoleglycerol-P (InGP) ? indole + glyceraldehyde-3-P, were determined for both the forward reaction (InGP to indole) and the reverse reaction (indole to InGP). The majority of the mutant α-subunits had <10% of the wild-type activity in the forward reaction, as expected since these mutant strains were selected for defects in this reaction. In contrast, the majority of these mutant enzymes had >50% of the wild-type activity in the reverse reaction. Several had 5 to 15% of wild-type specific activity in the forward reaction but 60 to 100% of wild-type specific activity in the reverse reaction. Spontaneous revertant strains, selected for their increased ability to catalyze the forward reaction effectively, contained α-subunits with the expected higher specific activities in the forward reaction but without parallel changes in the reverse reaction activity.     

Enzymatic formation and esterification of (S)-mandelonitrile

The (S)-selective hydroxynitrile lyase from Hevea brasiliensis (HbHNL) catalyzes the trans-cyanohydrin reaction (transcyanation). The equilibrium of this two-step reaction sequence is not favorable unless a large excess of acetone cyanohydrin (1) is used. Therefore, the coupling of this reaction with a follow-up reaction was investigated. It was established that the trans-cyanohydrin reaction could be performed in organic media, making it possible to couple it with a lipase-catalyzed acylation. Candida antarctica lipase B (CAL-B) shows a high selectivity (E=100) for (S)-mandelonitrile (4) and is, therefore, the ideal candidate for this type of multi-step one-pot reaction.     

Interfacial versus homogeneous enzymatic cleavage of mandelonitrile by hydroxynitrile lyase in a biphasic system

The question of an interfacial versus a homogeneous reaction is carefully addressed for the enzymatic biphasic cleavage of mandelonitrile to benzaldehyde by Prunus amygdalus hydroxynitrile lyase (pa-Hnl) (Hickel et al. [1999] Biotechnol Bioeng 36:425-436). Experimental evidence, including 1) the reaction ceases when the interface is populated by previously adsorbed denatured pa-Hnl, 2) the reaction continues even after washout of the bulk enzyme from the aqueous phase, 3) highly nonpolar organic solvents initially promote fast reaction kinetics that relatively quickly decay to zero product production, and 4) the reaction rate is nonlinear in the bulk enzyme concentration, provide robust grounds for an interfacial reaction. We also model enzymatic mandelonitrile cleavage assuming a homogeneous aqueous-phase reaction. The homogeneous reaction scheme does not simultaneously account for the experimental observations of a linear dependence of the reaction rate on organic/water interfacial area, no dependence on the aqueous-phase volume, and a nonlinear dependence on pa-Hnl aqueous concentration. Further, simple calculations demonstrate that the homogeneous reaction rate is at least three orders of magnitude slower than those observed by Hickel et al. (1999). We again conclude that enzyme adsorbed at the organic solvent/water interface primarily catalyzes the biphasic mandelonitrile cleavage reaction.     

Structure of Exocellular Polymers and Their Relationship to Bacterial Flocculation

Various bacteria which degrade pyruvate by the phosphoroclastic reaction were examined with respect to the role of coenzyme A (CoA) in this reaction. The strictly anaerobic bacteria, which cleave pyruvate by the phosphoroclastic reaction characteristic of Clostridia, required catalytic levels of CoA for the CO(2)-pyruvate exchange and acetoin-forming portions of the phosphoroclastic reaction. These reactions were reversibly inhibited by the CoA analogue, desulfo-CoA. In contrast, using cell-free extracts of bacteria which degrade pyruvate by the coliform phosphoroclastic reaction (pyruvate formate-lyase), no requirement for CoA could be observed for the formate-pyruvate exchange reaction. It is suggested that CoA serves a regulatory function in the early portion of the clostridal type of phosphoroclastic reaction.     

Biosynthesis of Chlorophyll of Photosystem II Reaction Centers in Plant Leaves in Early Stages of Etiolation

Spectral methods were used to study the sequences of chlorophyll biosynthesis reactions in etiolated pea, bean, and maize plants in early stages (3-4 days) of growth. For these juvenile plants, along with the reaction chain known for mature (7-9 day-old) plants, a new reaction chain was found which started with phototransformation of the long-wavelength form PChld 686/676 into PChld 653/648. (PChld 653/648 differs from the main known precursor form PChld 655/650). The subsequent photoreduction of PChld 653/648 leads to the formation of Chld 684/676, which is transformed into Chl 688/680 in the course of a dark reaction. After completion of this reaction, fast (20-30 sec) quenching of the fluorescence of the reaction product is observed with the formation of non-fluorescent Chl 680. The reaction accompanied by pigment fluorescence quenching is absent in pea mutants with depressed function of Photosystem II reaction centers. This suggests that the newly found reaction chain leads to the formation of chlorophyll of the Photosystem II reaction center.     

Candida rugosa lipase-catalyzed intramolecular O- to N- transacylation of butyryl propranolol in the presence of cyclodextrins

In the present paper, a novel enzymatic reaction between (R,S)-O-butyryl propranolol (O-BP) and lipase from Candida rugosa in the presence of hydroxypropyl-beta-cyclodextrin (HP-beta-CD) is described. Under the used condition, lipase catalyzed the intramolecular transacylation of O-BP into N-butyryl propranolol (N-BP). Propranolol, the product of the expected hydrolysis reaction, was not detected in the reaction medium. A chiral analysis of the reaction product indicated that lipase showed a preference for (R)-O-butyryl propranolol since it first transformed the (R)-enantiomer and then the corresponding (S)-enantiomer. The influence of different reaction conditions on the initial rate is also studied.     

Occurrence of early and late asthmatic reaction after provocation with antigen and the status of pulmonary ventilation in patients with hay fever after the pollination season]

Pulmonary ventilation and asthmatic reaction under laboratory conditions have been investigated in 23 patients with allergic rhinitis hypersensitive to grass pollen. Pulmonary ventilation has been assessed with the aid of VCin, FVC, FEV1, FEV1/VCin, PEF, MEF50, and MTT. Asthmatic reaction has been produced by an inhalation of allergens mixture with dose-response technique. An early reaction has been diagnosed, when FEV1 decreased by at least 20% or MEF50 by 30% within 10 minutes, and late reaction when the same parameters decreased after 6 or 24 hours. An early asthmatic reaction has been noted in 2 patients (8.7%), late--in 4 patients (17.4%), and double (both early and late) reaction in 2 patients (8.7%). Pulmonary ventilation has been normal in all examined patients, except two of them with peripheral airways obstruction (MEF50 less than 70% of the normal value). Results suggest, that asthmatic reaction may be provoked in the laboratory in patients with pollinosis and normal pulmonary ventilation after pollen season. Such a reaction may also be expected during a natural exposition to pollens.     

Characterization of Phosphoenolpyruvate Carboxykinase from Panicum maximum

Phosphoenolpyruvate carboxykinase, EC 4.1.1.32 (PEPCK), was purified 43-fold from the grass Panicum maximum. Michaelis constants (Km) were determined for the exchange reaction, the carboxylation reaction, and the decarboxylation reaction. The Km values for oxaloacetate and ATP in the decarboxylation reaction were found to be lower than the Km values for the substrates used in the exchange reaction and in the carboxylation reaction. Phosphoenolpyruvate carboxylase was not detectable in the purified PEPCK preparation.     

Examination of the reaction of fully reduced cytochrome oxidase with hydrogen peroxide by flow-flash spectroscopy

The reaction of cytochrome c oxidase with hydrogen peroxide has been of great value in generating and characterizing oxygenated species of the enzyme that are identical or similar to those formed during turnover of the enzyme with dioxygen. Most previous studies have utilized relatively low peroxide concentrations (millimolar range). In the current work, these studies have been extended to the examination of the kinetics of the single turnover of the fully reduced enzyme using much higher concentrations of peroxide to avoid limitations by the bimolecular reaction. The flow-flash method is used, in which laser photolysis of the CO adduct of the fully reduced enzyme initiates the reaction following rapid mixing of the enzyme with peroxide, and the reaction is monitored by observing the absorbance changes due to the heme components of the enzyme. The following reaction sequence is deduced from the data. (1) The initial product of the reaction appears to be heme a(3) oxoferryl (Fe(4+)=O(2)(-) + H(2)O). Since the conversion of ferrous to ferryl heme a(3) (Fe(2+) to Fe(4+)) is sufficient for this reaction, presumably Cu(B) remains reduced in the product, along with Cu(A) and heme a. (2) The second phase of the reaction is an internal rearrangement of electrons and protons in which the heme a(3) oxoferryl is reduced to ferric hydroxide (Fe(3+)OH(-)). In about 40% of the population, the electron comes from heme a, and in the remaining 60% of the population, Cu(B) is oxidized. This step has a time constant of about 65 micros. (3) The third apparent phase of the reaction includes two parallel reactions. The population of the enzyme with an electron in the binuclear center reacts with a second molecule of peroxide, forming compound F. The population of the enzyme with the two electrons on heme a and Cu(A) must first transfer an electron to the binuclear center, followed by reaction with a second molecule of peroxide, also yielding compound F. In each of these reaction pathways, the reaction time is 100-200 micros, i.e., much faster than the rate of reaction of peroxide with the fully oxidized enzyme. Thus, hydrogen peroxide is an efficient trap for a single electron in the binuclear center. (4) Compound F is then reduced by the final available electron, again from heme a, at the same rate as observed for the reduction of compound F formed during the reaction of the fully reduced oxidase with dioxygen. The product is the fully oxidized enzyme (heme a(3) Fe(3+)OH(-)), which reacts with a third molecule of hydrogen peroxide, forming compound P. The rate of this final reaction step saturates at high concentrations of peroxide (V(max) = 250 s(-)(1), K(m) = 350 mM). The data indicate a reaction mechanism for the steady-state peroxidase activity of the enzyme which, at pH 7.5, proceeds via the single-electron reduction of the binuclear center followed by reaction with peroxide to form compound F directly, without forming compound P. Peroxide is an efficient trap for the one-electron-reduced state of the binuclear center. The results also suggest that the reaction of hydrogen peroxide to the fully oxidized enzyme may be limited by the presence of hydroxide associated with the heme a(3) ferric species. The reaction of hydrogen peroxide with heme a(3) is very substantially accelerated by the availability of an electron on heme a, which is presumably transferred to the binuclear center concomitant with a proton that can convert the hydroxide to water, which is readily displaced.     

Theoretical studies on the Mo-catalyzed asymmetric intramolecular Pauson-Khand-type [2?+?2?+?1] cycloadditions of 3-allyloxy-1-propynylphosphonates

Density functional theory (DFT) was used to investigate the Mo-catalyzed intramolecular Pauson-Khand reaction of 3-allyloxy-1-propynylphosphonates. All intermediates and transition states were optimized completely at the B3LYP/6-31 G(d,p) level [LANL2DZ(f) for Mo]. In the Mo-catalyzed intramolecular Pauson-Khand reaction, the C–C oxidative cyclization reaction was the chirality-determining step, and the reductive elimination reaction was the rate-determining step. The carbonyl insertion reaction into the Mo–C(sp(3)) bondwas easier than into the Mo–C=C bond. And the dominant product predicted theoretically was of (S)-chirality, which agreed with experimental data. This reaction was solventd ependent, and toluene was the best among the three solvents toluene, CH3CN, and THF.     

Mechanism of dihydroneopterin aldolase. NMR, equilibrium and transient kinetic studies of the Staphylococcus aureus and Escherichia coli enzymes

Dihydroneopterin aldolase (DHNA) catalyzes both the cleavage of 7,8-dihydro-D-neopterin (DHNP) to form 6-hydroxymethyl-7,8-dihydropterin (HP) and glycolaldehyde and the epimerization of DHNP to form 7,8-dihydro-L-monapterin (DHMP). Whether the epimerization reaction uses the same reaction intermediate as the aldol reaction or the deprotonation and reprotonation of C2' of DHNP has been investigated by NMR analysis of the reaction products in a D2O solvent. No deuteration of C2' was observed for the newly formed DHMP. This result strongly suggests that the epimerization reaction uses the same reaction intermediate as the aldol reaction. In contrast with an earlier observation, the DHNA-catalyzed reaction is reversible, which also supports a nonstereospecific retroaldol/aldol mechanism for the epimerization reaction. The binding and catalytic properties of DHNAs from both Staphylococcus aureus (SaDHNA) and Escherichia coli (EcDHNA) were determined by equilibrium binding and transient kinetic studies. A complete set of kinetic constants for both the aldol and epimerization reactions according to a unified kinetic mechanism was determined for both SaDHNA and EcDHNA. The results show that the two enzymes have significantly different binding and catalytic properties, in accordance with the significant sequence differences between them.