A kinetic study of lipase-catalyzed reversible kinetic resolution involving verification at miniplant-scale

Lipase-catalyzed kinetic resolution of racemates is a popular method for synthesis of chiral synthons. Most of these resolutions are reversible equilibrium limited reactions. For the first time, an extensive kinetic model is proposed for kinetic resolution reactions, which takes into account the full reversibility of the reaction, substrate inhibition by an acyl donor and an acyl acceptor as well as alternative substrate inhibition by each enantiomer. For this purpose, the reversible enantioselective transesterification of (R/S)-1-methoxy-2-propanol with ethyl acetate catalyzed by Candida antarctica lipase B (CAL-B) is investigated. The detailed model presented here is valid for a wide range of substrate and product concentrations. Following model discrimination and the application of Haldane equations to reduce the degree of freedom in parameter estimation, the 11 free parameters are successfully identified. All parameters are fitted to the complete data set simultaneously. Six types of independent initial rate studies provide a solid data basis for the model. The effect of changes in substrate and product concentration on reaction kinetics is discussed. The developed model is used for simulations to study the behavior of reaction kinetics in a fixed bed reactor. The typical plot of enantiomeric excess versus conversion of substrate and product is evaluated at various initial substrate mixtures. The model is validated by comparison with experimental results obtained with a fixed bed reactor, which is part of a fully automated state-of-the-art miniplant.     

A kinetic model of the cytochrome bf complex. Evaluation of kinetic parameters

A kinetic model of the cytochrome bf complex was developed on the assumption that the Q-cycle operates. The bf complex was considered as a membrane enzyme catalyzing the electron transfer from plastoquinol to plastocyanine, which is coupled with proton translocation from the chloroplast stroma to the thylakoid lumen. The dependence of the electron transfer rates on the value of the transmembrane electric potential was taken into account. The model was applied to describe the experimental data on the flash-induced turnover of cytochromes b, plastocyanine, and the kinetics of proton deposition in the thylakoid lumen. The estimation of model parameters was performed.     

Dynamic kinetic resolution

The use of transition-metal-enzyme combinations to effect tandem in situ racemisation and resolutions has extended the scope of dynamic kinetic resolutions. This methodology has signalled a more proactive stance to the racemisation process, which has traditionally relied on more fortuitous approaches, namely the exploitation of labile substrates. The development and application of specific racemising enzymes such as mandelate racemase offers potential for future multienzyme dynamic kinetic resolutions.     

Multiscale kinetic analysis of proteins

The stochasticity of molecular motion results in the existence of multiple kinetically relevant pathways in many biomolecular mechanisms. Because it is highly demanding to characterize them for complex systems, mechanisms are often described with a single-pathway perspective. However, kinetic network analysis and sub-ensemble experimental insight are increasingly demonstrating not only the existence of competing pathways but also the importance of kinetic selection in biology. This review focuses on advances in multiscale kinetic analysis of proteins, which connects molecular level information from simulations to macroscopic data to characterize mechanistic reaction networks and the reactive flux through them. We describe a range of methods used and highlight several examples where kinetic modeling has revealed functional importance of pathway heterogeneity.     

The kinetic mechanism of kinesin

The chemical kinetic mechanism of kinesin (K) is considered by using a consensus scheme incorporating biochemically defined open, closed and trapped states. In the absence of microtubules, the dominant species is a trapped K*ADP state, which is defined by its ultra-slow release of ADP (off rate, k(off) approximately 0.002 s(-1)) and weak microtubule binding (dissociation constant, K(d) approximately 10-20 microM). Once bound, this trapped state equilibrates with a strongly binding open state that rapidly releases ADP (k(off) approximately 300 s(-1)). After ADP release, Mg*ATP binds (on rate, k(on) approximately 2 microM(-1)s(-1)) driving formation of a closed state that is defined by hydrolysis competence and by strong binding to microtubules. Hydrolysis (k(hyd) approximately 100-300 s(-1)) and phosphate release (k(off)>100 s(-1)) both occur in this microtubule-bound closed state. Phosphate release acts as a gate that controls reversion to the trapped K*ADP state, which detaches from the microtubule, completing the cycle.     

Transient kinetic studies support refinements to the chemical and kinetic mechanisms of aminolevulinate synthase

5-Aminolevulinate synthase catalyzes the pyridoxal 5'-phosphate-dependent condensation of glycine and succinyl-CoA to produce carbon dioxide, CoA, and 5-aminolevulinate, in a reaction cycle involving the mechanistically unusual successive cleavage of two amino acid substrate alpha-carbon bonds. Single and multiple turnover rapid scanning stopped-flow experiments have been conducted from pH 6.8-9.2 and 5-35 degrees C, and the results, interpreted within the framework of the recently solved crystal structures, allow refined characterization of the central kinetic and chemical steps of the reaction cycle. Quinonoid intermediate formation occurs with an apparent pK(a) of 7.7 +/- 0.1, which is assigned to His-207 acid-catalyzed decarboxylation of the alpha-amino-beta-ketoadipate intermediate to form an enol that is in rapid equilibrium with the 5-aminolevulinate-bound quinonoid species. Quinonoid intermediate decay occurs in two kinetic steps, the first of which is acid-catalyzed with a pK(a) of 8.1 +/- 0.1, and is assigned to protonation of the enol by Lys-313 to generate the product-bound external aldimine. The second step of quinonoid decay defines k(cat) and is relatively pH-independent and is assigned to opening of the active site loop to allow ALA dissociation. The data support important refinements to both the chemical and kinetic mechanisms and indicate that 5-aminolevulinate synthase operates under the stereoelectronic control predicted by Dunathan's hypothesis.     

Chemoenzymatic dynamic kinetic resolution

During the past decade a new concept has appeared in asymmetric catalysis involving the combination of a biocatalyst and a chemocatalyst in one 'pot' leading to efficient deracemization via dynamic kinetic resolution (DKR). Here, we outline the different strategies that have been developed for efficient chemoenzymatic DKR, in particular the powerful combination of an enzyme and a metal catalyst.     

Mycobacterium tuberculosis mycothione reductase: pH dependence of the kinetic parameters and kinetic isotope effects

The recent identification of the enzyme in Mycobacterium tuberculosis that catalyzes the NADPH-dependent reduction of the unique low molecular weight disulfide mycothione, mycothione reductase, has led us to examine the mechanism of catalysis in greater detail. The pH dependence of the kinetic parameters V and V/K for NADPH, NADH, and an active analogue of mycothione disulfide, des-myo-inositol mycothione disulfide, has been determined. An analysis of the pH profiles has allowed the tentative assignment of catalytically significant residues crucial to the mechanism of disulfide reduction, namely, the His444-Glu449 ion pair and Cys39. Solvent kinetic isotope effects were observed on V and V/K(DIMSSM), yielding values of 1.7 +/- 0.2 and 1.4 +/- 0.2, respectively, but not on V/K(NADPH). Proton inventory studies (V versus mole fraction of D(2)O) were linear, indicative of a single proton transfer in a solvent isotopically sensitive step. Steady-state primary deuterium kinetic isotope effects on V have been determined using NADPH and NADH, yielding values of 1.27 +/- 0.03 and 1.66 +/- 0.14, respectively. The pre-steady-state primary deuterium kinetic isotope effect on enzyme reduction has values of 1.82 +/- 0.04 and 1.59 +/- 0.06 for NADPH and NADH, respectively. The steady-state primary deuterium kinetic isotope effect using NADH coincide with that obtained under single turnover conditions, suggesting the complete expression of the intrinsic primary kinetic isotope effect. Rapid reaction studies on the reductive half-reaction using NADPH and NADH yielded maximal rates of 129 +/- 2 and 20 +/- 1 s(-1), respectively, while similar studies of the oxidation of the two-electron reduced enzyme by mycothiol disulfide yielded a maximum rate of 190 +/- 10 s(-1). These data suggest a unique flavoprotein disulfide mechanism in which the rate of the oxidative half-reaction is slightly faster than the rate of the reductive half-reaction.     

Process kinetic analysis of pleuromulin fermentation

The industrial production of pleuromulin, an example for the production of secondary metabolites, has been carried out of the corporation Biochemie Kundl/Austria. The results of the experiments, especially the process kinetics are analyzed following the principles of the integrating strategy, which has been developed by the authors.Experiments on technical and laboratory scale were carefully designed and evaluated in order to allow an adequate insight in to this complex bioprocess. Typical plots are presented to illustrate the general macroscopic behaviour which serve as a reliable basis for process quantification.Mathematic modelling of this bioprocess will be presented in a following paper.List of Symbols n l/min rotational speed - P kg/m³ pleuromulin concentration - q _P h^–1 specific production rate - q _S1 h^–1 specific rate of glucose consumption - q _S2 h^–1 specific rate of oil consumption - r _P kg/m³ · h product formation rate - r _S1 kg/m³ · h glucose consumption rate - r _X kg/m³ · h growth rate - S ₁ kg/m³ glucose concentration - S ₂ kg/m³ oil concentration - T °C temperature - t h, d time - _g m³/h gas flow rate - X kg/m³ mycelium dry weight - Y _i/j yield coefficient - _app mPa s apparent viscosity - h^–1 specific growth rate The authors extend their gratitude to Biochemie Kundl Gmbh, where the experimental work has been carefully carried out     

Steady-state kinetic mechanism of PDK1

PDK1 catalyzes phosphorylation of Thr in the conserved activation loop region of a number of its downstream AGC kinase family members. In addition to the consensus sequence at the site of phosphorylation, a number of PDK1 substrates contain a PIF sequence (PDK1-interacting fragment), which binds and activates the kinase domain of PDK1 (PDK1(deltaPH)). To gain further insight to PIF-dependent catalysis, steady-state kinetic and inhibition studies were performed for His6-PDK1(deltaPH)-catalyzed phosphorylation of PDK1-Tide (Tide), which contains an extended "PIF" sequence C-terminal to the consensus sequence for PDK1 phosphorylation. In two-substrate kinetics, a large degree of negative binding synergism was observed to occur on formation of the active ternary complex (alphaKd(ATP) = 40 microM and alphaKd(Tide) = 80 microM) from individual transitory binary complexes (Kd(ATP) = 0.6 microM and Kd(Tide) = 1 microM). On varying ATP concentrations, the ADP product and the (T/E)-PDK1-Tide product analog (p'Tide) behaved as competitive and noncompetitive inhibitors, respectively; on varying Tide concentrations, ADP and p'Tide behaved as noncompetitive and competitive inhibitors, respectively. Also, negative binding synergism was associated with formation of dead-end inhibited ternary complexes. Time progress curves in pre-steady-state studies under "saturating" or kcat conditions showed (i) no burst or lag phenomena, (ii) no change in reaction velocity when adenosine 5'-O-(thiotriphosphate) was used as a phosphate donor, and (iii) no change in reaction velocity on increasing relative microviscosity (0 < or = eta/eta0 < or = 3). Taken together, PDK1-catalyzed trans-phosphorylation of PDK1-Tide approximates a Rapid Equilibrium Random Bi Bi system, where motions in the central ternary complex are largely rate-determining.     

Discrete design of enzyme kinetic experiments

The usefulness of discrete designs in enzyme kinetics as an alternative to continuous designs is discussed in this paper, focusing on designs satisfying the D-optimality criterion. This study has been carried out using a program called DODID, specifically devised for this purpose, which is available by request to the authors. The results presented in this paper show that the relative efficiency of the D-optimal discrete designs with respect to the continuous ones increases rapidly when increasing the number of possible values for the control variables. Relative efficiencies higher than 0.98 are achieved when using 20 possible values for each variable. The power of the tools provided by the computational approach of this work is proved by the analysis made on the robustness of different designs for estimating the kinetic parameters when a wrong assumption on the error structure has been made. The robustness of the designs made assuming medium constant error (error variance proportional to the true response) is thus confirmed. A comparative study of several discriminating designs is also presented. The results obtained show that the designs produced by adding the optimal discrete designs corresponding to both candidate models plus the point where the weighted difference between the predicted values is maximum, is a good choice when designing an experiment for discrimination.