Continuous enzymatic production of peptide precursor in aqueous/organic biphasic medium

N-(benzyloxycarbonyl)-L-aspartic acid (Z-L-Asp) has generally been used as a carboxyl substrate for the enzymatic synthesis of a precursor of aspartame (synthetic sweetener); however, alternative inexpensive protection groups have been in demand for lowering the total cost of its industrial-scale production. A formyl group (F-) was found to be a more desirable protecting group for the N-terminus of amino acid derivatives due to its low cost of preparation, introduction, and removal. The yield of F-AspPheOMe (N-formyl-L-aspartyl-L-phe- nylalanine methylester), however, was found to be <10% in a conventional aqueous medium. We found that F-L-Asp and L-PheOMe were partitioned mainly to the aqueous phase in an aqueous/organic biphasic medium, whereas F-AspPheOMe partitioned to the organic phase, especially when some extracting agents were added. In this study, simultaneous operation of an enzymatic reaction and a product separation by liquid-liquid extraction was thus applied to the F-AspPheOMe synthesis. We succeeded in synthesizing F-AspPheOMe continuously in an aqueous/tributylphosphate (TBP) biphasic medium with >95% yield, which was about tenfold higher than that in an aqueous monophasic medium.     

Reaction mechanism of non-allosteric phosphofructokinase.

The reaction mechanism of the non-allosteric phosphofructokinase from Lactobacillus plantarum was investigated by initial-rate bisubstrate kinetics and product inhibition kinetics adn by the measurement of equilibrium isotope exchange in the presence of various substrate and product concentrations. The reaction mechanism is clearly sequential. The product inhibition and equilibrium isotope-exchange patterns are consistent with an ordered bi-bi reaction sequence with fructose 6-phosphate as the leading substrate and ADP as the first product released from the enzyme.     

Internal thermodynamics of enzymes determined by equilibrium quench: values of Kint for enolase and creatine kinase

The equilibrium constant (Kint) for the enzyme-bound substrate and product of a one substrate/one product enzyme (enolase) and for those of a two substrate/two product enzyme (creatine kinase) have been determined. The values of Kint were determined by the rapid quenching of equilibrium mixtures of enzyme and radiolabeled substrate and product, under conditions where all of the marker substrate and product are bound. The scope and limitations of this method are discussed. Values of Kint have been collected from the literature, and it is shown that these data are consistent with the theory for kinetically optimized enzymes that is developed in the preceding paper.     

Synthesis of aspartame precursor by solid thermolysin in organic solvent

Summary The enzymatic synthesis of a peptide compound was carried out successfully in homogeneous organic solvent.Solid Thermolysin was found to catalyze the synthetic reaction of N-benzyloxycarbonyl-L-aspartyl-L-phenylalanine methyl ester (Z-APM; a precursor of sweetner Aspartame) from N-benzyloxycarbonyl-L-aspartic acid (Z-L-Asp) and L-phenylalanine methyl ester (L-PheOMe) in a 98 percent organic medium (ethylacetatebenzenemethanolwater=5029192). The dissolution of enzyme was not observed. The optimal pH shifted to acidic side by 1.0 pH unit, compared with that in aqueous medium. The enzymatic activity of solid thermolysin with an average size of 3.4×9.5 m was determined to be 0.18 moles-product/(mg-solid)·h under the initial concentrations of L-PheOMe of 0.1M and Z-L-Asp of 0.05M, and at pH 6.0 and 40°C.     

Steady state and equilibrium exchange kinetic studies of the sheep brain glutamine synthetase reaction.

The kinetic mechanism of the sheep brain glutamine synthetase has been examined by both initial rate kinetics using the glutamate analog beta-glutamate and by isotope exchange measurements at equilibrium. Results of the initial rate studies were compatible with a number of sequential mechanisms but not with a partially or fully ordered rapid equilibrium or a ping-pong mechanism. Kinetic parameters at 37 degrees and pH 7.2 were K beta-Glu = 16 mM, KATP = 0.28 mM, and KNH2OH = 1.4 mM. For all equilibrium exchanges studied (ATP in equilibrium ADP, ATP in equilibrium Pi, and Glu in equilibrium Gln), the rate of exchange rose smoothly to a maximum as all substrates and products were simultaneously raised in a constant ratio. This result is in accord with a random order of substrate addition. A brief treatment of equilibrium exchange rates in cases where all substrate/product pairs are varied together is also presented.     

Kinetics of substrate recognition and cleavage by human 8-oxoguanine-DNA glycosylase

Human 8-oxoguanine-DNA glycosylase (hOgg1) excises 8-oxo-7,8-dihydroguanine (8-oxoG) from damaged DNA. We report a pre-steady-state kinetic analysis of hOgg1 mechanism using stopped-flow and enzyme fluorescence monitoring. The kinetic scheme for hOgg1 processing an 8-oxoG:C-containing substrate was found to include at least three fast equilibrium steps followed by two slow, irreversible steps and another equilibrium step. The second irreversible step was rate-limiting overall. By comparing data from Ogg1 intrinsic fluorescence traces and from accumulation of products of different types, the irreversible steps were attributed to two main chemical steps of the Ogg1-catalyzed reaction: cleavage of the N-glycosidic bond of the damaged nucleotide and β-elimination of its 3′-phosphate. The fast equilibrium steps were attributed to enzyme conformational changes during the recognition of 8-oxoG, and the final equilibrium, to binding of the reaction product by the enzyme. hOgg1 interacted with a substrate containing an aldehydic AP site very slowly, but the addition of 8-bromoguanine (8-BrG) greatly accelerated the reaction, which was best described by two initial equilibrium steps followed by one irreversible chemical step and a final product release equilibrium step. The irreversible step may correspond to β-elimination since it is the very step facilitated by 8-BrG.     

The binding of substrates and a product of the enzymatic reaction to glutathione S-transferase A.

The binding of substrates and a product to glutathione S-transferase A from rat liver was studied by use of equilibrium dialysis and equilibrium partition in a two-phase system. The radioactive substrates glutathione and bromosulfophthalein as well as a product of glutathione and 3,4-dichloro-1-nitrobenzene, S-(2-chloro-4-nitrophenyl)glutathione, gave hyperbolic binding isotherms with a stoichiometry of 2 mol per mol of enzyme (i.e. 1 molecule per subunit). Glutathione (and glutathione disulfide) had an equilibrium (dissociation) constant for the binding of about 10 microM, whereas bromosulfophthalein and the product had equilibrium constants of about 0.5 microM. All ligands showed the same binding stoichiometry, and competition experiments involving unlabeled ligands indicated that glutathione and the glutathione derivatives were binding to the same site. Low affinity sites appeared to exist in addition to the specific high affinity sites (one per subunit) for all ligands tested. The binding studies are fully consistent with a steady state random kinetic mechanism for the enzyme.     

Equilibrium shifts in enzyme reactions at high pressure

The effect of pressure on the equilibrium of a reaction was studied. Theoretical equilibrium constants and product concentrations have been calculated at elevated pressures. The theory is illustrated with an example of l-malate synthesis catalyzed by a fumarase. To study shifts in the equilibrium relatively low pressures can be applied (50–200 MPa), but our calculations show that for process optimisation much higher pressures (up to 1000 MPa) have to be used.

At these higher pressures, more stable enzymes are needed. We performed experiments with the hyperthermophilic β-glycosidase from Pyrococcus furiosus as a catalyst. Oligosaccharides were synthesized from glucose in an equilibrium reaction at pressures from 0.1 to 500 MPa. The enzyme remained active at 500 MPa. The equilibrium of the reaction was influenced by pressure and shifted towards the hydrolysis side, decreasing final oligosaccharide concentrations with increasing pressure. This pressure dependence of the final product concentration and the equilibrium constant could be described with a positive reaction volume of 2.4 mol/cm³.     

The horseradish peroxidase-catalyzed oxidation of 3,5,3',5'-tetramethylbenzidine. Free radical and charge-transfer complex intermediates

Benzidine and related compounds are well known substrates for horseradish peroxidase/H2O2 oxidation. Typically, two different colored products are formed. In this paper, we study the oxidation of 3,5,3',5'-tetramethylbenzidine. The first colored product is a blue charge-transfer complex of the parent diamine and the diimine oxidation product. This species exists in rapid equilibrium with the radical cation. The radical was observed by ESR spectroscopy, and hyperfine splitting constants were determined. Addition of equimolar hydrogen peroxide yields the yellow diimine, which is stable at acid pH. At less than equimolar peroxide, all four species (diamine, radical cation, charge-transfer complex, and diimine) exist in equilibrium. A theoretical analysis of this redox system is presented, including a determination of the extinction coefficients and equilibrium constant for the nonradical species.     

Hydrogen regulation of acetogenesis from glucose by freely suspended and immobilised acidogenic cells in continuous culture

Summary The pattern of end product formation from glucose was examined in an acidogenic culture where the hydrogen partial pressure was controlled by headspace gas recirculation and nitrogen purging. The results demonstrate that physical control of hydrogen displacement can shift the equilibrium toward acetate production with a concomitant fall in reduced end product formation in the freely-suspended cell system. Immobilised acidogenic cell did not show the same pattern of end product formation when subject to reduction in hydrogen partial pressure which has implications for control of methanogenic end product in two stage anaerobic digester systems using immobilised cell technology.     

Kinetic behavior of slowly equilibrating association-dissociation enzyme systems]

The shape of the plots of product accumulation versus time (t) has been analysed for slowly equilibrating association-dissociation enzyme systems of the types 2p in equilibrium P (P is enzyme oligomer which is able to dissociate reversibly forming two identical halves p) and M in equilibrium M2 in equilibrium M2 in equilibrium... (M is monomer which has two association sites overlapping with active sites). It is assumed that the rate of equilibration between oligomeric forms is comparable with the rate of over-all enzymatic reaction and that substrate-oligomer complexes are in rapid equilibrium with free components. It has been shown that characteristic feature of kinetic behavior of slowly equilibrating association-dissociation enzyme systems is that the value of tau depends on enzyme concentration (tau is the intercept on t-axis for linear asymptota of the curve of product concentration versus time at t leads to infinity).     

Metabolically driven equilibrium shift of asymmetric amination of ketones by ω-transaminase using alanine as an amino donor

Removal of a side product to overcome unfavorable equilibrium is a prerequisite for the asymmetric amination of ketones using ω-transaminase (ω-TA). Alanine has been preferred as an amino donor because its deamination product (i.e. pyruvate) is easily removable by several enzymatic methods. Here, we demonstrated that the removal of pyruvate by an innate metabolic pathway could afford equilibrium shift of the ω-TA reactions.     

Influence of substrate and product diffusion on the heterogeneous kinetics of enzymic reversible reactions

When a reversible reaction is catalyzed by a surface bound enzyme, the diffusion of both substrate and product can considerably modify the kinetic properties of the reaction. According to this theoretical analysis, the enzyme activity is decreased due to the presence of substrate and product concentration gradients in the enzyme microenvironment, and the relative kinetic importance of the two diffusion steps mainly depend on the value of a dimensionless criterion inversely proportional to the equilibrium constant. Moreover diffusional effects increase with increasing bound enzyme activity, but decrease with increasing substrate and product concentration. Analytical expressions are established for the limiting values of substrate and product concentrations in the enzyme microenvironment, as well as for the increase in half-maximal-activity substrate concentration in the presence of substrate and product diffusional limitations.     

Kinetics and equilibrium of enzymatic synthesis of peptides in aqueous/organic biphasic systems. Thermolysin-catalyzed synthesis of N-(benzyloxycarbonyl)-L-aspartyl-L-phenylalanine methyl ester

We studied kinetics and the equilibrium relationship for the thermolysin-catalyzed synthesis of N-(benzyloxycarbonyl)-L-aspartyl-L-phenylalanine methyl ester (Z-Asp-PheOMe) from N-(benzyloxycarbonyl)-L-aspartic acid (Z-Asp) and L-phenylalanine methyl ester (PheOMe) in an aqueous-organic biphasic system. This is a model reaction giving a condensation product with dissociating groups. The kinetics for the synthesis of Z-Asp-PheOMe in aqueous solution saturated with ethyl acetate was expressed by a rate equation for the rapid-equilibrium random bireactant mechanism, and the reverse hydrolysis reaction was zero-order with respect to Z-Asp-PheOMe concentration. The courses of synthesis of Z-Asp-PheOMe in the biphasic system were well explained, by the rate equations obtained for the aqueous solution and by the partition of substrate and condensation product between the both phases. The rate of synthesis in the biphasic system was much lower than in aqueous solution due to the unfavorable partition of PheOMe in the aqueous phase. The equation for the equilibrium yield of Z-Asp-PheOMe in the biphasic system was derived assuming that only the non-ionized forms of the substrate and condensation product exist in the organic phase. It was found theoretically and experimentally that the yield of Z-Asp-PheOMe is maximum at the aqueous-phase pH of around 5, lower than for synthesis in aqueous solution. The effect of the organic solvent on the rate and equilibrium for the synthesis of Z-Asp-PheOMe could be explained by the variation in the partition coefficient. The effect of the partitioning of substrate on the aqueous-phase pH change was also shown.     

An investigation of basic assumption in enzyme kinetics using results of the geometric theory of differential equations

Up to the present time, the following property of the product component in the reversible one substrate-one intermediate-one product enzymic mechanism has been taken only as anassumption, viz., during the course of the reaction, the time-rate of change of product concentration is never negative and the product concentration never exceeds its equilibrium value. Applying the methods of the geometric theory of ordinary differential equations it is shown that this result follows as a direct deduction from the differential equations governing the mechanism together with the initial conditions. Further, the nature of the equilibrium point as a stable node is established.     

Steady state and exchange kinetics of pyruvate, phosphate dikinase from Propionibacterium shermanii.

Evidence is presented based on requirements for exchange in the partial reactions, initial velocity and exchange kinetics and product inhibition, that the pyruvate, phosphate dikinase reaction of propionibacteria occurs by a nonclassical Tri Uni Uni Ping Pong mechanism. The mechanism involves a pyrophosphoryl enzyme, a phosphoryl enzyme, and the free enzyme, and three functionally distinct and independent substrate sites. On the first site, there is pyrophosphorylation of the enzyme by ATP with subsequent release of AMP. The pyrophosphoryl moiety then reacts at the second site with Pi yielding the product PPi and the phosphoryl from of the enzyme. At the third site pyruvate is phosphorylated yielding P-enolpyruvate and the free enzyme. The three catalytic sites are proposed to be linked by a histidyl residue which functions as a pyrophosphoyrl- and phosphoryl-carrier between the three sites. This proposal is based on the following observations. (A) The patterns of the double reciprocal plots of the initial velocities were all parallel; (b) product inhibition between each pair of substrates and products of the three partial reactions were competitive, i.e. ATP against AMP, Pi against PPi, and pyruvate against P-enolpyruvate; (c) the other product inhibitions, with one exception, were noncompetitive as required by the nonclassical ping-pong mechanism; (d) ATP or P-enolpyruvate was required for the Pi in equilibrium PPi exchange reaction which is in accord with the participation of a pyrosphosphoryl or phosphoryl form of the enzyme in this exchange; (e) the ATP in equilibrium AMP exchange and pyruvate in equilibrium P-enolpyruvate exchange did not require additional substrates. In addition, the inhibition and participation in the exchange reactions of the alpha,beta and beta,gamma-methylene analogues of ATP and of the methylene analogue of inorganic pyrophosphate were investigated and the results were in accord with the proposed mechanism. The combined evidence provides a well documented example of a three site nonclassical Tri Uni Uni Ping Pong mechanism.     

Production of 4-valerolactone by an equilibrium-limited transformation in a partitioning bioreactor: impact of absorptive polymer properties

The biotransformation of levulinic acid to 4-valerolactone (4VL) is pH-dependent and equilibrium limited, distinct from the more common irreversible biotransformations that are constrained by product toxicity or biocatalyst inhibition. Our processing strategy for this system was to selectively remove the product, 4VL, which is in equilibrium with its precursor, 4-hydroxyvalerate (4HV), to pull the reaction to a greater extent of conversion. 4VL is challenging to separate from the aqueous phase due to its water miscibility, necessitating the use of water-absorbing polymers to provide affinity toward the hydrophilic product. Manipulating the composition of copolymers, thereby varying the architecture of polymer chains, conferred drastically different extents of water absorption and caused different biotransformation outcomes. A custom-synthesized random copolymer designed to maximize the proportion of material with affinity for the solute had high water uptake, which resulted in the poor selectivity for the target molecule relative to its precursor. Conversely, a moderately water-absorbing commercial segmented block copolymer, Hytrel^® 8206, demonstrated selectivity toward 4VL relative to its precursor, 4HV, and increased 4VL production by approximately 30 % by shifting the equilibrium toward the product. This work has shown that water absorption is an important, previously neglected criterion in evaluating polymer affinity and selectivity toward hydrophilic target molecules.     

A rapid equilibrium random sequential bi-bi mechanism for human placental glutathione S-transferase

Double-reciprocal plots of initial-rate data for the conjugation of 1-chloro-2,4-dinitrobenzene (CDNB) and GSH by human placental GSH S-transferase pi were linear for both substrates. Computer modelling of the initial-rate data using nonlinear least-squares regression analysis favoured a rapid equilibrium random sequential bi-bi mechanism, over a steady-state random sequential mechanism or a steady-state or rapid equilibrium ordered mechanism. KGSH was calculated as 0.125 +/- 0.006 mM, KCDNB was 0.87 +/- 0.07 mM and alpha was 2.1 +/- 0.3 for the rapid equilibrium random model. The product, S-(2,4-dinitrophenyl)glutathione, was a competitive inhibitor with respect to GSH, and a mixed-type inhibitor toward CDNB (KP = 18 +/- 3 microM). The observed pattern of inhibition is consistent with a rapid equilibrium random mechanism, with a dead-end enzyme.CDNB.product complex, but inconsistent with the inhibition patterns of other bireactant mechanisms. Since rat liver GSH S-transferase 3-3 acts via a steady-state random sequential mechanism [1], while human placental GSH S-transferase and perhaps also rat liver GSH S-transferase 1-1 [2] exhibit rapid equilibrium random mechanisms, we conclude that the kinetic mechanism of the GSH S-transferases is isoenzyme-dependent.     

Prochiral selectivity and intramolecular isotope effects in the cytochrome P-450 catalyzed omega-hydroxylation of cumene

A kinetic model is presented from which steady-state equations are derived that describe the intramolecular competition for the enzymatically mediated hydroxylation of two like groupings of a prochiral substrate. The observed isotope effect in such a system if one of the groupings is isotopically labeled is shown to be a function of three parameters: the equilibrium constant for the catalytically sensitive orientations of the two prochiral groupings at the active site, the intrinsic isotope effect associated with the bond-breaking step, and the relative rates of bond breaking vs. enzyme-substrate dissociation. The expected isotope effects associated with the omega-hydroxylation of racemic, (R)-, and (S)-2-phenylpropane-1,1,1-d3 and the product stereoselectivity associated with the omega-hydroxylation of (R)- and (S)-[1-13C]-2-phenylpropane were determined with microsomal preparations (cytochrome P-450) from untreated and phenobarbital- and beta-naphthoflavone-pretreated male Sprague-Dawley rats. The data from these experiments allow the observed isotope effect to be evaluated in terms of its component parts, i.e., expected isotope effects, product stereoselectivity, and equilibrium constant. These data further suggest that the intramolecular isotope effect is consistent with a hydrogen abstraction recombination mechanism and is largely dependent upon the chemical nature of the porphyrin-Fe-oxene complex but independent of specific apoprotein structure, product stereoselectivity is primarily dependent upon apoprotein structure, and product stereoselectivity is a good measure of the equilibrium constant and both parameters are dependent upon the chirality of the active site.     

Formation of stable anhydrides from CoA transferase and hydroxamic acids.

Acetohydroxamic acid reacts with the enzyme-CoA form of succinyl-CoA:3-ketoacid coenzyme A transferase to give an inactive product with a rate constant of 860 M-1 min-1 at pH 8.1, 25 degrees C. The reaction is reversible in the presence of coenzyme A and has an equilibrium constant of 0.040. The product is an anhydride that is an analog of the intermediate that has been postulated in the normal catalytic pathway; it is inactive because coenzyme A does not react with the acyl group of the hydroxamic acid. The equilibrium constant for formation of the anhydride from the thil ester of enzyme and methyl 3-mercaptopropionate is 75 times larger than the equilibrium constant of 2.2 for the formation of N,O-diacetylhydroxylamine from acetohydroxamic acid and acetyl-CoA. This shows that the enzyme stabilizes the anhydride at the active site by at least -2.6 kcal mol-1. Succinomonohydroxamic acid reacts with enzyme-CoA as both a substrate and an inactivator, with relative rate constants of 25:1. The inactivation is irreversible, indicating that the enzyme provides a larger stabilization of at least -5.9 kcal mol-1 for the anhydride of an analog of the specific substrate, succinate. The results are consistent with the hypothesis that the enzyme stabilizes an anhydride that is formed at the active site during turnover of normal substrates through a stepwise reaction mechanism.