Kinetics of suicide substrates

In the light of the scheme presented by Walsh et al. (1978, Meth. Enzymol. 53, 437-448), Waley (1980, Biochem. J. 185, 771-773) and Tatsunami et al. (1981, Biochim. biophys. Acta, 662, 226-235) made a detailed study for the kinetics of suicide substrates. However, the effects of the enzyme reaction intermediate and product were not considered in their studies. Equations for the kinetics of suicide substrate were derived for a more reasonable scheme. A plotting method was proposed to determine the kinetic parameters and it has been shown that suicide substrate can be used to study the formative mechanism of enzyme-catalyzed reaction intermediate.     

A kinetic study of the suicide inactivation of an enzyme measured through coupling reactions. Application to the suicide inactivation of tyrosinase.

A systematic procedure for the kinetic study of reaction mechanisms with enzyme inactivation induced by a suicide substrate in the presence or in the absence of an auxiliary substrate, when the enzyme activity is measured through coupling reactions, enzymically catalysed or not, was developed and analysed by using the transient-phase approach. The methodology is established to determine the parameters and kinetic constants corresponding to the enzyme suicide inactivation and the coupling reactions. This approach is illustrated by a study of the suicide inactivation of tyrosinase by catechol in the presence of L-proline. Treatment of the experimental data was carried out by non-linear regression.     

Cytochrome P-450-catalyzed dehydrogenation of 1,4-dihydropyridines

A variety of different 4-substituted 1,4-dihydropyridine Hantzsch esters are substrates for ring dehydrogenation by a cytochrome P-450 (P-450) enzyme (P-450 UT-A); the substitutent could be varied from a hydrogen to a naphthalenyl, but a pyrenyl derivative was not dehydrogenated. When a 4-alkyl group is present, both the P-450 which oxidizes the substrate and other P-450s can be inactivated (by putative alkyl radicals). P-450s did not discriminate with regard to removal of the 4-H atoms from an enantiomeric pair of dihydropyridines. Losses of the 4-proton and N-methyl from a N-methyl-1,4-dihydropyridine occur at similar rates. The calculated intrinsic kinetic hydrogen isotope effect (Dk) for dehydrogenation of 1,4-dihydro-2,6-dimethyl-4-phenyl-3,5-pyridinedicarboxylic acid dimethyl ester was 2.9 in a reconstituted P-450 UT-A enzyme system. No significant kinetic hydrogen isotope effect was observed in microsomal incubations for the dehydrogenation of this compound or 1,4-dihydro-2,6-dimethyl-3,5-pyridinedicarboxylic acid diethyl ester in a variety of competitive and noncompetitive experiments. In light of previous studies on the magnitude of kinetic hydrogen isotope effects in P-450 systems (e.g. Miwa et al., 1983 (Miwa, G. T., Walsh, J. S., Kedderis, G. L., and Hollenberg, P. F. (1983) J. Biol. Chem. 258, 14445-14449], the mechanistic proposals of Augusto et al., 1982 (Augusto, O., Beilan, H. S., and Ortiz de Montellano, P. R. (1982) J. Biol. Chem. 257, 11288-11295)) for enzyme inactivation by 4-alkyl-substituted Hantzsch pyridine esters, and other precedents for sequential electron transfer in amine oxidation by P-450s, we interpret these results as being consistent with P-450-mediated 1-electron oxidation of dihydropyridines followed by the facile loss of the 4-proton, with subsequent electron transfer to complete the reaction.     

Recognition of damaged DNA by Escherichia coli Fpg protein: insights from structural and kinetic data

Formamidopyrimidine-DNA glycosylase (Fpg) excises oxidized purines from damaged DNA. The recent determination of the three-dimensional structure of the covalent complex of DNA with Escherichia coli Fpg, obtained by reducing the Schiff base intermediate formed during the reaction [Gilboa et al., J. Biol. Chem. 277 (2002) 19811] has revealed a number of potential specific and non-specific interactions between Fpg and DNA. We analyze the structural data for Fpg in the light of the kinetic and thermodynamic data obtained by the method of stepwise increase in ligand complexity to estimate relative contributions of individual nucleotide units of lesion-containing DNA to its total affinity for this enzyme [Ishchenko et al., Biochemistry 41 (2002) 7540]. Stopped-flow kinetic analysis that has allowed the dissection of Fpg catalysis in time [Fedorova et al., Biochemistry 41 (2002) 1520] is also correlated with the structural data.     

Enzyme kinetics by mid-infrared spectroscopy: β-fructosidase study by a one-step assay

An alternate method for enzyme study is proposed. Multidimensional statistical analysis applied on mid-infrared attenuated total reflectance spectra (Cadet et al. (1991) Appl. Spectrosc. 42, 166–172) collected during a kinetic allows a direct and fast quantification of the remaining substrate, as well as a one step enzymatic assay. Furthermore, the combination of these techniques may be used as a structural tool. The method applied to the study of β-fructosidase is developed in this paper as an example. With appropriate calibration, the method may be extend to any enzyme.     

An automated method for acetylcholinesterase kinetics

An automated assay for acetylcholinesterase (EC 3.1.1.7.) has been developed based on the manual spectrophotometric method of Ellmanet al. (1). This method was used to determine (a) the enzyme activity of an unknown sample and (b) the dependence of initial rates given by a fixed enzyme concentration on the substrate concentration. Methods to minimize possible enzyme modification by DTNB (2) are described. Finally a modification of the conventional autoanalyser procedure permitted rapid and reproducible enzyme kinetic analysis under various conditions. This helped to minimize the effects of possible enzyme inactivation at high dilutions especially when using crude enzyme preparations.     

Investigation of kinetics of immobilized liver esterase by flow calorimetry

Flow calorimetry (FC) was shown to be a powerful tool for investigation of the kinetics of phenyl acetate hydrolysis catalyzed by pig liver carboxyl esterase. The enzyme was immobilized in alginate gel particles that were placed in a calorimetric flow column and the heat effect of enzyme reaction was followed in single flow and total recirculation conditions. It was shown that the registered temperature change was proportional to molar amount of substrate transformed in the column. A mathematical model describing the enzyme reaction, mass transfer, and heat effects in the calorimetric system was developed and used for the kinetic data evaluation. By combining data from single flow and recirculation modes true kinetic parameters were evaluated by the proposed mathematical procedure based on the model solution and successive approximations.

The kinetic data for carboxyl esterase showed a slide substrate inhibition by phenyl acetate. The obtained kinetic parameters were as follows: Michaelis constant K_m=2 mmol dm⁻³ and substrate inhibition constant K_i=42 mmol dm⁻³. The method can be applied to kinetic study of immobilized enzymes directly in the flow calorimeter without any requirement of an independent analytical technique.     

A kinetic model for enzyme interfacial activity and stability: pa-hydroxynitrile lyase at the diisopropyl ether/water interface

A kinetic framework is developed to describe enzyme activity and stability in two-phase liquid-liquid systems. In particular, the model is applied to the enzymatic production of benzaldehyde from mandelonitrile by Prunus amygdalus hydroxynitrile lyase (pa-Hnl) adsorbed at the diisopropyl ether (DIPE)/aqueous buffer interface (pH = 5.5). We quantitatively describe our previously obtained experimental kinetic results (Hickel et al., 1999; 2001), and we successfully account for the aqueous-phase enzyme concentration dependence of product formation rates and the observed reaction rates at early times. Multilayer growth explains the early time reversibility of enzyme adsorption at the DIPE/buffer interface observed by both enzyme-activity and dynamic-interfacial-tension washout experiments that replace the aqueous enzyme solution with a buffer solution. The postulated explanation for the unusual stability of pa-Hnl adsorbed at the DIPE/buffer interface is attributed to a two-layer adsorption mechanism. In the first layer, slow conformational change from the native state leads to irreversible attachment and partial loss of catalytic activity. In the second layer, pa-Hnl is reversibly adsorbed without loss in catalytic activity. The measured catalytic activity is the combined effect of the deactivation kinetics of the first layer and of the adsorption kinetics of each layer. For the specific case of pa-Hnl adsorbed at the DIPE/buffer interface, this combined effect is nearly constant for several hours resulting in no apparent loss of catalytic activity. Our proposed kinetic model can be extended to other interfacially active enzymes and other organic solvents. Finally, we indicate how interfacial-tension lag times provide a powerful tool for rational solvent selection and enzyme engineering.     

Determination of elasticities, concentration and flux control coefficients from transient metabolite data using linlog kinetics

This paper presents a practical approach to estimate the kinetic parameters of a metabolic network from in vivo kinetics experiments. This method is based on the linlog kinetics format (Visser and Heijnen, 2003, Metab. Eng. 5(3), 164-176; Wu et al., 2004, Eur. J. Biochem. 271, 3348-3359), of which the kinetic parameters, called elasticities, are estimated by an iterative linear optimization followed by non-linear optimization, from transient metabolite concentration data which are directly obtainable from rapid pulse experiments. In this way, not only the parameters are estimated but also a full kinetic model, based on linlog kinetics, is developed. The obtained elasticities also allow immediate calculation of all control coefficients. As an in silico case study, the estimation of elasticities of a linear pathway is presented. The method is shown to be able to estimate the elasticities quite accurately and to be robust toward errors in the metabolite data originating from sampling and measurement inaccuracy. The method allows experimental redesign to get more accurate estimated parameters and accommodates various types of experimentally applied disturbances in the pathway: changes in independent metabolites, dependent metabolites or enzyme levels/activities.     

Suicide-peroxide inactivation of horseradish peroxidase in the presence of sodium n-dodecyl sulphate: a study of the enzyme deactivation kinetics

In the presence of the anionic surfactant sodium n-dodecyl sulphate (SDS), horseradish peroxidase (HRP) undergoes a deactivation process. Suicide inactivation of horseradish peroxidase by hydrogen peroxide(3 mM) was monitored by the absorbance change in product formation in the catalytic reaction cycle. The progress curve of the catalytic reaction cycle was obtained at 27degrees C and phosphate buffer 2.5 mM (pH = 7.0). The corresponding kinetic parameters i.e., intact enzyme activity (alpha i); the apparent rate constant of suicide inactivation by peroxide (ki); and the apparent rate constants of enzyme deactivation by surfactant (kd) were evaluated from the obtained kinetic equations. The experimental data are accounted for by the equations used in this investigation. Addition of SDS to the reaction mixture intensified the inactivation process. The deactivation ability of denaturant could be resolved from the observed inactivation effect of the suicide substrate by applying the proposed model. The results indicate that the deactivation and the inactivation processes are independent of each other.     

Modeling intrinsic kinetics of enzymatic cellulose hydrolysis

A multistep approach was taken to investigate the intrinsic kinetics of the cellulase enzyme complex as observed with hydrolysis of noncrystalline cellulose (NCC). In the first stage, published initial rate mechanistic models were built and critically evaluated for their performance in predicting time-course kinetics, using the data obtained from enzymatic hydrolysis experiments performed on two substrates: NCC and alpha-cellulose. In the second stage, assessment of the effect of reaction intermediates and products on intrinsic kinetics of enzymatic hydrolysis was performed using NCC hydrolysis experiments, isolating external factors such as mass transfer effects, physical properties of substrate, etc. In the final stage, a comprehensive intrinsic kinetics mechanism was proposed. From batch experiments using NCC, the time-course data on cellulose, cello-oligosaccharides (COS), cellobiose, and glucose were taken and used to estimate the parameters in the kinetic model. The model predictions of NCC, COS, cellobiose, and glucose profiles show a good agreement with experimental data generated from hydrolysis of different initial compositions of substrate (NCC supplemented with COS, cellobiose, and glucose). Finally, sensitivity analysis was performed on each model parameter; this analysis provides some insights into the yield of glucose in the enzymatic hydrolysis. The proposed intrinsic kinetic model parametrized for dilute cellulose systems forms a basis for modeling the complex enzymatic kinetics of cellulose hydrolysis in the presence of limiting factors offered by substrate and enzyme characteristics.     

Kinetic analysis of enzyme systems with suicide substrate in the presence of a reversible competitive inhibitor, tested by simulated progress curves

The use of suicide substrates remains a very important and useful method in enzymology for studying enzyme mechanisms and designing potential drugs. Suicide substrates act as modified substrates for the target enzymes and bind to the active site. Therefore the presence of a competitive reversible inhibitor decreases the rate of substrate-induced inactivation and protects the enzyme from this inactivation. This lowering on the inactivation rate has evident physiological advantages, since it allows the easy acquisition of experimental data and facilitates kinetic data analysis by providing another variable (inhibitor concentration). However despite the importance of the simultaneous action of a suicide substrate and a competitive reversible inhibition, to date no corresponding kinetic analysis has been carried out. Therefore we present a general kinetic analysis of a Michaelis-Menten reaction mechanism with double inhibition caused by both, a suicide substrate and a competitive reversible inhibitor. We assume rapid equilibrium of the reversible reaction steps involved, while the time course equations for the reaction product have been derived with the assumption of a limiting enzyme. The goodness of the analytical solutions has been tested by comparison with the simulated curves obtained by numerical integration. A kinetic data analysis to determine the corresponding kinetic parameters from the time progress curve of the product is suggested. In conclusion, we present a complete kinetic analysis of an enzyme reaction mechanism as described above in an attempt to fill a gap in the theoretical treatment of this type of system.     

Kinetic study in the transient phase of the suicide inactivation of frog epidermis tyrosinase

This paper deals with the kinetic study of a multisubstrate mechanism with enzyme inactivation induced by a suicide substrate. A transient phase approach has been developed that enables the deduction of explicit equations of product concentration vs. time. From these equations kinetic constants which characterize the suicide substrate can be obtained. This study with tyrosinase enzyme, which acts on L-dopa and catechol allowed us to determine the corresponding kinetic parameters, indicating that catechol is about 8-times more powerful as a suicide substrate than is L-dopa.     

Mechanism-based inactivation of VanX, a D-alanyl-D-alanine dipeptidase necessary for vancomycin resistance

VanX is a zinc-dependent D-Ala-D-Ala amino dipeptidase required for high-level resistance to vancomycin. The enzyme is also able to process dipeptides with bulky C-terminal amino acids [Wu, Z., Wright, G. D., and Walsh, C. T. (1995) Biochemistry 34, 2455-2463]. We took advantage of this observation to design and synthesize the dipeptide-like D-Ala-D-Gly(SPhip-CHF(2))-OH (7) as a potential mechanism-based inhibitor. VanX-mediated peptide cleavage generates a highly reactive 4-thioquinone fluoromethide which is able to covalently react with enzyme nucleophilic residues, resulting in irreversible inhibition. Inhibition of VanX by 7 was time-dependent (K(irr) = 30+/-1 microM; k(inact) = 7.3+/- 0.3 min(-1)) and active site-directed, as deduced from substrate protection experiments. Nucleophilic compounds such as sodium azide, potassium cyanide, and glutathione did not protect the enzyme from inhibition, indicating that the generated nucleophile inactivates VanX before leaving the active site. The failure to reactivate the dead enzyme by gel filtration or pH modification confirmed the covalent nature of the reaction that leads to inactivation. Inactivation was associated with the elimination of fluoride ion as deduced from (19)F NMR spectroscopy analysis and with the production of fluorinated thiophenol dimer 12. These data are consistent with suicide inactivation of VanX by dipeptide 7. The small size of the VanX active site and the presence of a number of nucleophilic side chains at the opening of the active site gorge [Bussiere, D. E., et al. (1998) Mol. Cell 2, 75-84] associated with the high observed partition ratio of 7500+/-500 suggest that the inhibitor is likely to react at the entrance of the active site cavity.     

Enantioselective synthesis of (S)-ibuprofen ester prodrug in cyclohexane by Candida rugosa lipase immobilized on Accurel MP1000

An enantioselective esterification process was developed for the synthesis of 2-N-morpholinoethyl (S)-ibuprofen ester prodrug from racemic ibuprofen by using Candida rugosa lipase immobilized on Accurel MP1000 in cyclohexane. Compared with the performance of Lipase MY, the immobilized lipase possesses a higher enzyme activity and thermal stability, but with a slightly suppressed enantioselectivity. A kinetic model was proposed and confirmed from experiments, for the simulation of time-course conversions of both enantiomers at various combinations of substrate concentrations in a batch reactor. Preliminary results of employing the proposed model and the immobilized lipase in a continuous packed-bed reactor were also reported and discussed.     

Dynamics of anaerobic growth of Saccharomyces cerevisiae in the chemostat.

A kinetic Monod model has been used to describe the dynamic response of a continuous stirred tank fermentor (CSTF) to changes in dilution rate. A general analytical solution of a linearized model was obtained. Experimental results (Vairo et al. 1977) of continuous anaerobic culture of Saccharomyces cerevisiae have verified the model quantitatively. For step disturbances on the dilution rate the responses of biomass concentration and the outlet substrate concentration were calculated on a digital computer and compared with the experimental data.     

A mathematical model of sialylation of N-linked oligosaccharides in the trans-Golgi network

A mathematical model is developed of the compartmentalized sialylationof N-linked oligosaccharides in order to understand and predictthe outcome of sialylation reactions. A set of assumptions arepresented, including Michaelis-Menten-type dependency of reactionrate on the concentration of the glycoprotein substrate. Theresulting model predicts the heterogeneous outcome of a posttranslationaloligosaccharide biosynthesis step, a critical aspect that isnot accounted for in the modeling of the cotranslational attachmentof oligosaccharides to glycosylation sites (Shelikoff et al.,Biotech. Bioeng., 50, 73–90, 1996) or general models ofthe secretion process (Noe and Delenick, J. Cell Sci, 92, 449–459,1989). In the steady-state for the likely case where the concentrationof substrate is much less than the K_m of the sialyltransferase,the model predicts that the extent of sialylation, x, will dependupon the enzyme concentration, enzyme kinetic parameters andsubstrate residence time in the reaction compartment. The valueof x predicted by the model using available literature datais consistent with the values of x that have been recently determinedfor the glycoproteins CD4 (Spellman et al, Biochemistry, 30,2395–2406, 1991) and t-PA (Spellman et al, J. Biol Chem.,264, 14100–14111, 1989) secreted by Chinese hamster ovarycells. For the unsaturated case, the model also predicts thatx is independent of the concentration of secreted glycoproteinin the Golgi. The general modeling approach outlined in thisarticle may be applicable to other glycosylation reactions andposttranslational modifications. model sialylation N-linked glycosylation     

Studies on structure-function relationships of indolepyruvate decarboxylase from Enterobacter cloacae, a key enzyme of the indole acetic acid pathway.

Enterobacter cloacae, isolated from the rhizosphere of cucumbers, produces large amounts of indole-3-acetic acid. Indolepyruvate decarboxylase, the key enzyme in the biosynthetic pathway of indole-3-acetic acid, catalyses the formation of indole-3-acetaldehyde and carbon dioxide from indole-3-pyruvic acid. The enzyme requires the cofactors thiamine diphosphate and magnesium ions for catalytic activity. Recombinant indolepyruvate decarboxylase was purified from the host Escherichia coli strain JM109. Specificity of the enzyme for the substrates indole-3-pyruvic acid, pyruvic acid, benzoylformic acid, and seven benzoylformic acid analogues was investigated using a continuous optical assay. Stopped-flow kinetic data showed no indication for substrate activation in the decarboxylation reaction of indole-3-pyruvic acid, pyruvic acid or benzoylformic acid. Size exclusion chromatography and small angle X-ray solution scattering experiments suggested the tetramer as the catalytically active state and a pH-dependent subunit association equilibrium. Analysis of the kinetic constants of the benzoylformic acid analogues according to Hansch et al. [Hansch, C., Leo, A., Unger, S.H., Kim, K.H., Nikaitani, D & Lien, E.J. (1973) J. Med. Chem.16, 1207-1216] and comparison with indole-3-pyruvic acid conversion by pyruvate decarboxylases from Saccharomyces cerevisiae and Zymomonas mobilis provided some insight into the catalytic mechanism of indolepyruvate decarboxylase.     

Dynamics of tyrosine hydroxylase mediated regulation of dopamine synthesis

Tyrosine hydroxylase's catalysis of tyrosine to dihydroxyphenylalanine (DOPA) is the highly regulated, rate-limiting step catalyzing the synthesis of the catecholamine neurotransmitter dopamine. Phosphorylation, cofactor-mediated regulation, and the cell's redox status, have been shown to regulate the enzyme's activity. This paper incorporates these regulatory mechanisms into an integrated dynamic model that is capable of demonstrating relative rates of dopamine synthesis under various physiological conditions. Most of the kinetic equations and substrate parameters used in the model correspond with published experimental data, while a few which were not available in literature have been optimized based on explicit assumptions. This kinetic pathway model permits a comparison of the relative regulatory contributions made by variations in substrate, phosphorylation, and redox status on enzymatic activity and permits predictions of potential disease states. For example, the model correctly predicts the recent observation that individuals with haemochromatosis and having excessive iron accumulation are at increased risk for acquiring Parkinsonism, a defect in neuronal dopamine synthesis (Bartzokis et al., 2004; Costello et al., 2004). Alpha synuclein mediated regulation of tyrosine hydroxylase has also been incorporated in the model, allowing an insight into the over-expression and aggregation of alpha synuclein in Parkinson's disease. Action Editor: Upinder Bhalla     

Kinetic analysis of enzyme systems with suicide substrate in the presence of a reversible, uncompetitive inhibitor.

We present a general kinetic analysis of enzyme catalyzed reactions evolving according to a Michaelis-Menten mechanism, in which an uncompetitive, reversible inhibitor acts. Simultaneously, enzyme inactivation is induced by an unstable suicide substrate, i.e. it is a Michaelis-Menten mechanism with double inhibition: one originating from the substrate and another originating from the reversible inhibitor. Rapid equilibrium of the reversible reaction steps involved is assumed and the time course equations for the reaction product have been derived under the assumption of limiting enzyme. The goodness of the analytical solutions has been tested by comparison with simulated curves obtained by numerical integration. A kinetic data analysis to determine the corresponding kinetic parameters from the time progress curve of the product is suggested.