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1.
The kinetic behaviour of insect acetylcholinesterases deviates from the Michaelis-Menten pattern. These deviations are known as activation or inhibition at various substrate concentrations and can be more or less observable depending on mutations around the active site of the enzyme. Most kinetic studies on these enzymes still rely on initial rate measurements. It is demonstrated here that according to this method one of the deviations can be overlooked. We attempt to point out that in such cases a detailed step-by-step progress curves analysis is successful. The study is focused on two different methods of analysing progress curves: (i) the first one is based on an integrated initial rate equation which can sufficiently fit truncated progress curves under corresponding conditions; and (ii) the other one precludes the algebraic formulae, but uses numerical integration for searching a non analytical solution of ordinary differential equations describing a kinetic model. All methods are tested on three different acetylcholinesterase mutants from Drosophila melanogaster. The results indicate that kinetic parameters for the E107K mutant with highly expressive activation and inhibition can be well evaluated applying any analysis method. It is quite different for E107W and E107Y mutants where latent activation is present, but discovered only using one or the other progress curves analysis methods.  相似文献   

2.
The time course of the residual enzyme activity of a general model consisting of an autocatalytic zymogen activation process inhibited by an irreversible competitive inhibitor and an irreversible uncompetitive inhibitor has been studied. Approached analytical expressions which furnish the time course of the residual enzyme activity from the onset of the reaction depending on the rate constants and initial concentration have been obtained. The goodness and limitations of the analytical equations were checked by comparing with the results obtained from the numerical integration, i.e. with the simulated progress curves. A dimensionless parameter giving the relative contributions of both the activation and the inhibitions routes is suggested, so that the value of this parameter determines whether the activation or the inhibitions routes prevail or if both processes are balanced during the time for which the analytical expressions are valid. The effects of the initial zymogen, free enzyme and inhibitors concentrations are analysed. Finally an experimental design and kinetic data analysis is proposed to evaluate simultaneously the kinetic parameters involved and to discriminate between different zymogen activation processes which can be considered particular cases of the general model.  相似文献   

3.
The time course of the residual enzyme activity of a general model consisting of an autocatalytic zymogen activation process inhibited by an irreversible competitive inhibitor and an irreversible uncompetitive inhibitor has been studied. Approached analytical expressions which furnish the time course of the residual enzyme activity from the onset of the reaction depending on the rate constants and initial concentration have been obtained. The goodness and limitations of the analytical equations were checked by comparing with the results obtained from the numerical integration, i.e. with the simulated progress curves. A dimensionless parameter giving the relative contributions of both the activation and the inhibitions routes is suggested, so that the value of this parameter determines whether the activation or the inhibitions routes prevail or if both processes are balanced during the time for which the analytical expressions are valid. The effects of the initial zymogen, free enzyme and inhibitors concentrations are analysed. Finally an experimental design and kinetic data analysis is proposed to evaluate simultaneously the kinetic parameters involved and to discriminate between different zymogen activation processes which can be considered particular cases of the general model.  相似文献   

4.
The kinetics of glyoxalase I [(R)-S-lactoylglutathione methylglyoxal-lyase; EC 4.4.1.5] and glyoxalase II (S-2-hydroxyacylglutathione hydrolase; EC 3.1.2.6) from Saccharomyces cerevisiae was studied in situ, in digitonin permeabilized cells, using two different approaches: initial rate analysis and progress curves analysis. Initial rate analysis was performed by hyperbolic regression of initial rates using the program HYPERFIT. Glyoxalase I exhibited saturation kinetics on 0.05-2.5 mM hemithioacetal concentration range, with kinetic parameters Km 0.53 +/- 0.07 mM and V (3.18 +/- 0.16) x 10(-2) mM.min(-1). Glyoxalase II also showed saturation kinetics in the SD-lactoylglutathione concentration range of 0.15-3 mM and Km 0.32 +/- 0.13 mM and V (1.03 +/- 0.10) x 10(-3) mM.min(-1) were obtained. The kinetic parameters of both enzymes were also estimated by nonlinear regression of progress curves using the raw absorbance data and integrated differential rate equations with the program GEPASI. Several optimization methods were used to minimize the sum of squares of residuals. The best parameter fit for the glyoxalase I reaction was obtained with a single curve analysis, using the irreversible Michaelis-Menten model. The kinetic parameters obtained, Km 0.62 +/- 0.18 mM and V (2.86 +/- 0.01) x 10(-2) mM.min(-1), were in agreement with those obtained by initial rate analysis. The results obtained for glyoxalase II, using either the irreversible Michaelis-Menten model or a phenomenological reversible hyperbolic model, showed a high correlation of residuals with time and/or high values of standard deviation associated with Km. The possible causes for the discrepancy between data obtained from initial rate analysis and progress curve analysis, for glyoxalase II, are discussed.  相似文献   

5.
The rate equation for a tight-binding inhibitor of an enzyme-catalysed first-order reversible reaction was used to derive two integrated equations. One of them covers the situations in which competitive, uncompetitive or non-competitive inhibition occurs and the other refers to the special non-competitive case where the two inhibition constants are equal. For these equations, graphical and non-linear regression methods are proposed for distinguishing between types of inhibition and for calculating inhibition constants from progress-curve data. The application of the non-linear regression to the analysis of stimulated progress curves in the presence of a tight-binding inhibitor is also presented. The results obtained are valid for any type of 'dead-end'-complex-forming inhibitor and can be used to characterize an unknown inhibitor on the basis of progress curves.  相似文献   

6.
(S)-Hydroxynitrile lyase (EC 4.1.2.39) from Hevea brasiliensis(rubber tree) catalyzes the reversible cleavage of cyanohydrins to aldehydes or ketones and prussic acid (HCN). Enzyme kinetics in both directions was studied on a model system with mandelonitrile, benzaldehyde, and HCN using two different methods-initial rate measurements and progress curve analysis. To discriminate between possible mechanisms with the initial rate method, product inhibition was studied. Benzaldehyde acts as a linear competitive inhibitor against mandelonitrile whereas HCN shows S-linear I-parabolic mixed-type inhibition. These results indicate an Ordered Uni Bi mechanism with the formation of a dead-end complex of enzyme, (S)-mandelonitrile and HCN. Prussic acid is the first product released from the enzyme followed by benzaldehyde. For progress curve analysis, a kinetic model of an Ordered Uni Bi mechanism including a dead-end complex, enzyme inactivation, and the chemical parallel reaction was set up, which described the experimental values very well. From the reaction rates obtained the kinetic constants were calculated and compared with the ones obtained from the initial rate method. Good agreement could be achieved between the two methods supporting the suggested mechanism. Copyright 1999 John Wiley & Sons, Inc.  相似文献   

7.
In the present paper, a kinetic analysis of a general model for proenzyme activation, where the activating enzyme and also the activated one are reversibly inhibited in two steps by two different inhibitors, has been performed. The cases in which both inhibitors are the same, or in which the inhibition is irreversible (only one or the two inhibition routes) are treated as particular cases of the general model. In addition, the kinetic behaviour of many other proenzyme activation systems involving inhibition, particular cases of the reaction scheme under study, can be obtained. The total number of particular cases for the general model under study is 370, so this approach offers to the scientific community working in limited proteolysis regulation for the first time a method based on general solutions which only needs to be specified to their concrete problem of zymogen activation. Finally, new adimensional parameters are introduced, allowing the knowledgement, in the case that any of the inhibition routes is irreversible, the relative weight of both activation and irreversible inhibition routes.  相似文献   

8.
Zhang Z  Huong SM  Wang X  Huang DY  Huang ES 《Journal of virology》2003,77(23):12660-12670
Previous work has demonstrated that the human cytomegalovirus IE1-72 protein is able to bind to the N terminus of p107, and IE1-72 alone is sufficient for alleviation of p107-mediated cell growth suppression. However, the mechanism of this alleviation is unclear. Here, we show that IE1-72 can alleviate p107 inhibition of cyclin E/cdk2 kinase activity. We cotransfected various IE1-72 and p107 constructs into C33A cells and demonstrated that IE1-72 could activate the kinase activity of cyclin E/cdk2. Conversely, IE2-86 did not activate this activity, suggesting that the interaction between p107 and IE1-72 and the subsequent kinase activation are specific. By the use of a series of deletion and point mutants of IE1-72 and p107, we observed that a mutation of the loop region of helix-loop-helix-turn-helix in exon 3 of IE1-72 as well as a mutation of the leucine zipper-2 region in exon 4 of IE1-72 abolished binding to p107. In addition, these two IE1-72 mutants did not alleviate p107 inhibition of cyclin E/cdk2 kinase activity and also failed to alleviate p107 inhibition of the E2F-responsive promoter. Meanwhile, deletion of the N-terminal aa 1 to 175 of p107 abolished both p107 binding with IE1-72 and p107 inhibition of cyclin E/cdk2 kinase activity. This result confirms that the N-terminus aa 1 to 175 region of p107 is a common region where both IE1-72 protein and cyclin E/cdk2 bind. We propose a mechanism in which binding of IE1-72 to p107 displaces cyclin E/cdk2 from p107. Once released from p107, cyclin E/cdk2 is able to function as an active kinase.  相似文献   

9.
The polymerization kinetics of sickle cell hemoglobin are found to exhibit stochastic variations when observed in very small volumes (approximately 10(-10) cm3). The distribution of progress curves has been measured at several temperatures for a 4.50 mM-hemoglobin S sample using a laser-photolysis, light-scattering technique. The progress curves at a given temperature are superimposable when translated along the time axis, showing that the variability of the kinetic progress curves results primarily from fluctuations in the time at which polymerization is initiated. The shapes of the initial part of the progress curves are well-fitted using the functional form I(t) = Io + As exp (Bt), derived from a dual nucleation model. When the distribution of the measured tenth times is broad, the rate of homogeneous nucleation can be obtained by fitting the exponential tail of the distribution. As the distribution sharpen, the rate of homogeneous nucleation can be estimated by modelling the width of the distribution function using a simple Monte-Carlo simulation of the polymerization kinetics. Using the rates of homogeneous nucleation obtained from the distributions, the rates of heterogeneous nucleation and polymer growth can be obtained from the experimental parameters As and B. The resulting nucleation rates are roughly 1000 times greater than those obtained from an analysis of bulk kinetic data. The results provide strong support for the dual-nucleation mechanism and show that the distribution of progress curves provides a powerful independent method for measuring the rate of homogeneous nucleation and thereby obtaining values for the other principal rates of the mechanism.  相似文献   

10.
1. Several methods of analysing progress curves of enzyme-catalysed reactions are discussed briefly in relation to their usefulness in a situation where a reaction product has a K(i) much lower than the K(m) for the substrate 2. A comparison is made of different methods of estimating initial rates in this situation. 3. The use of a computer curve-fitting routine capable of handling functions of more than one variable for the extraction of kinetic parameters from progress curves is described. 4. This method and that of fitting time as a polynomial in product concentration are applied to progress curves for the soluble mitochondrial adenosine triphosphatase and the results are compared with values obtained by more conventional methods.  相似文献   

11.
Sergienko EA  Jordan F 《Biochemistry》2002,41(12):3952-3967
Pyruvate decarboxylase from yeast (YPDC, EC 4.1.1.1) exhibits a marked lag phase in the progress curves of product (acetaldehyde) formation. The currently accepted kinetic model for YPDC predicts that, only upon binding of substrate in a regulatory site, a slow activation step converts inactive enzyme into the active form. This allosteric behavior gives rise to sigmoidal steady-state kinetics. The E477Q active site variant of YPDC exhibited hyperbolic initial rate curves at low pH, not consistent with the model. Progress curves of product formation by this variant were S-shaped, consistent with the presence of three interconverting conformations with distinct steady-state rates. Surprisingly, wild-type YPDC at pH < or =5.0 also possessed S-shaped progress curves, with the conformation corresponding to the middle steady state being the most active one. Reexamination of the activation by substrate of wild-type YPDC in the pH range of 4.5-6.5 revealed two characteristic transitions at all pH values. The values of steady-state rates are functions of both pH and substrate concentration, affecting whether the progress curve appears "normal" or S-shaped with an inflection point. The substrate dependence of the apparent rate constants suggested that the first transition corresponded to substrate binding in an active site and a subsequent step responsible for conversion to an asymmetric conformation. Consequently, the second enzyme state may report on "unregulated" enzyme, since the regulatory site does not participate in its generation. This enzyme state utilizes the alternating sites mechanism, resulting in the hyperbolic substrate dependence of initial rate. The second transition corresponds to binding a substrate molecule in the regulatory site and subsequent minor conformational adjustments. The third enzyme state corresponds to the allosterically regulated conformation, previously referred to as activated enzyme. The pH dependence of the Hill coefficient suggests a random binding of pyruvate in a regulatory and an active site of wild-type YPDC. Addition of pyruvamide or acetaldehyde to YPDC results in the appearance of additional conformations of the enzyme.  相似文献   

12.
Most enzyme kinetic experiments are carried out under pseudo-first-order conditions, that is, when one of the reactant species (the enzyme or the substrate) is in a large excess of the other species. More accurate kinetic information about the system can be gained without the restrictions of the pseudo-first-order conditions. We present a practical and general method of analysis of the common two-step rapid equilibrium Michaelis-Menten mechanism. The formalism is exact in that it does not involve any other approximations such as the steady-state, limitations on the reactant concentrations or on reaction times. We apply this method to the global analysis of kinetic progress curves for bovine alkaline phosphatase assays carried out under both pseudo-first-order and pseudo-second-order conditions.  相似文献   

13.
Case A  Huskey WP  Stein RL 《Biochemistry》2003,42(16):4727-4732
Investigating the selectivity that an enzyme expresses toward its substrates can be technically challenging if reaction of these substrates is not accompanied by a conveniently monitored change in some physicochemical property. In this paper, we describe a simple method for determining steady-state kinetic parameters for enzymatic turnover of such "silent" substrates. According to this method, silent substrate S is allowed to compete for enzymic reaction with signal-generating substrate S*, whose conversion to product can be conveniently monitored. Full reaction progress curves are collected under conditions of [S*](o) < K(m)* and [S](o) >or= 3K(m). Progress curves collected under these conditions are characterized by an initial lag phase of duration tau that is followed by the pseudo-first-order reaction of S. Steady-state kinetic parameters for the silent substrate can be obtained by one of two methods. One method combines least-squares fitting with numerical integration of appropriate rate equations to analyze the progress curves, while the other method relies on direct graphical analysis in which K(m) is the value of [S](o) that reduces the control velocity by a factor of 2 and V(max) is shown to simply equal the ratio [S](o)/tau. We use these methods to analyze the alpha-chymotrypsin-catalyzed hydrolysis of silent substrate Suc-Ala-Phe-AlaNH(2) with signal generator Suc-Ala-Phe-pNA. From the curve-fitting method, k(c) = 0.9 +/- 0.2 s(-1) and K(m) = 0.4 +/- 0.1 mM, while by direct graphical analysis, k(c) = 1.1 +/- 0.1 s(-1) and K(m) = 0.51 +/- 0.03 mM. As validation of this new method, we show agreement of these values with those determined independently by HPLC analysis of the hydrolysis of Suc-Ala-Phe-AlaNH(2) by alpha-CT, where k(c) = 1.1 +/- 0.1 s(-1) and K(m) = 0.5 +/- 0.1 mM.  相似文献   

14.
利用TG-DTG技术测得槲皮素在氮气气氛中不同升温速率β下的热分解曲线,协同使用Achar法、Coats-Redfern法、Kissinger法和Ozawa法等4种方法同时进行动力学处理,根据热分解的表观活化能Eα和指前因子A计算推断槲皮素的贮存期.随着升温速率的提高,槲皮素的热分解温度逐渐升高;槲皮素热二步分解的机理依次是随机成核与随后生长控制和化学反应控制,对应的函数名称是Avrami-Erofeev方程和反应级数方程;经Gaussian模拟和热重数据结合分析,槲皮素在第一步分解时,失去两个O原子;第二步分解时失去一个苯环;根据第一步热分解的表观活化能Eα和指前因子A推断,在室温25℃下,槲皮素的贮存期为1.5 ~2年.  相似文献   

15.
A global kinetic analysis of a model consisting of an autocatalytic zymogen-activation process, in which an irreversible inhibitor competes with the zymogen for the active site of the proteinase, and a monitoring coupled reaction, in which the enzyme acts upon one of its substrates, is presented. This analysis is based on the progress curves of any of the two products released in the monitoring reaction. The general solution is applied to an important particular case in which rapid equilibrium conditions prevail. Finally, we suggest a procedure to predict whether the inhibition or activation route dominates in the steady state of the system. These results generalize our previous analysis of simpler mechanisms.  相似文献   

16.
A method is described for quantifying the proteolytic action of the milk-clotting enzyme chymosin on small and medium-sized peptide substrates by monitoring the decrease of absorbance at 230 nm during cleavage. The method is illustrated by the determination of the kinetic parameters of the specific splitting of a kappa-casein-related hexa- and pentadecapeptide by chymosin. The results are in good agreement with those found earlier with the same enzyme/substrate system by using an automated ninhydrin method. Erroneous results were obtained when the kinetic data were derived from one single progress curve. The significance of initial rate measurements for calculating correct kinetic parameters is briefly discussed. The usefulness of single progress curves measured at different initial substrate concentrations for obtaining information about the mechanism of the enzymic reaction is demonstrated.  相似文献   

17.
The kinetics of heavy metal ions inhibition of jack bean urease was studied by progress curve analysis in a reaction system without enzyme-inhibitor preincubation. The inhibition was found to be biphasic with an initial, small inhibitory phase changing over the time course of 5-10 min into a final linear steady state with a lower velocity. This time-dependent pattern was best described by mechanism B of slow-binding inhibition, involving the rapid formation of an EI complex that subsequently undergoes slow conversion to a more stable EI* complex. The kinetic parameters of the process, the inhibition constants Ki and Ki* and the forward k5 and reverse k6 rate constants for the conversion, were evaluated from the reaction progress curves by nonlinear regression treatment. Based on the values of the overall inhibition constant Ki*, the heavy metal ions were found to inhibit urease in the following decreasing order: Hg2+ > Cu2+ > Zn2+ > Cd2+ > Ni2+ > Pb2+ > Co2+ > Fe3+ > As3+. With the Ki* values as low as 1.9 nM for Hg2+ and 7.1 nM for Cu2+, 100-1000 times lower than those of the other ions, urease may be utilized as a bioindicator of the trace levels of these ions in environmental monitoring, bioprocess control or pharmaceutical analysis.  相似文献   

18.
Interleukin 3 (IL-3) or granulocyte macrophage colony-stimulating factor (GM-CSF) activates c-fos, c-jun, and c-myc genes and proliferation in both hematopoietic and nonhematopoietic cells. Using a series of deletion mutants of the beta subunit of human GM-CSF receptor (hGMR) and inhibitors of tyrosine kinase, two distinct signaling pathways, one for activation of c-fos and c-jun genes, and the other for cell proliferation and activation of c-myc gene have been elucidated. In contrast to wealth of information on the pathway leading to activation of c-fos/c-jun genes, knowledge of the latter is scanty. To clarify the mechanisms of activation of c-myc gene by cytokines, we established a transient transfection assay in mouse proB cell line BA/F3 cells expressing hGMR. Analyses of hGMR beta subunit mutants revealed two cytoplasmic regions involved in activation of the c-myc promoter, one is essential and the other is dispensable but enhances the activity. These regions are located at the membrane proximal and the distal regions covering amino acid positions 455-544 and 544-589, respectively. Characterization of cis-acting regulatory elements of the c-myc gene showed that the region containing the P2 promoter initiation site is sufficient to mediate the response to mIL-3 or hGM-CSF. Electrophoretic mobility shift assay using an oligonucleotide corresponding to the distal putative E2F binding site revealed that p107/E2F complex, the negative regulator of E2F, decreased, and free E2F increased after mIL-3 stimulation. These results support the thesis that mIL-3 or hGM-CSF regulates the c-myc promoter by altering composition of the E2F complexes at E2F binding site.  相似文献   

19.
20.
Liu HY  Wang Z  Regni C  Zou X  Tipton PA 《Biochemistry》2004,43(27):8662-8669
Phosphomannomutase/phosphoglucomutase occupies a central position in the pathways by which several virulence factors are synthesized in Pseudomonas aeruginosa. Virtual screening was used to identify potential inhibitors of phosphomannomutase/ phosphoglucomutase, and one compound, the anthraquinone-based dye Disperse Blue 56, showed potent inhibition in vitro. The kinetics of inhibition was complex; the time courses for reactions in the presence of the inhibitor were biphasic, suggestive of slow-binding inhibition. Quantitative analysis of the progress curves and preincubation experiments demonstrated that slow-binding inhibition was not occurring, however. Initial velocity kinetic studies indicated that Disperse Blue 56 was a parabolic, noncompetitve inhibitor. Progress curves for reactions in the presence of Disperse Blue 56 could be fitted very well by a model in which 2 equiv of the inhibitor bound to free enzyme or the enzyme-substrate complex. The inhibition was largely relieved by the inclusion of 0.01% Triton X-100 in the assay solutions, which has been suggested to be the hallmark for inhibition by compounds that exert their effect through aggregates [McGovern, S. L., Caselli, E., Grigorieff, N., and Shiochet, B. K. (2002) J. Med. Chem. 45, 1712-1722]. Our kinetic data appear to be consistent with either inhibition by a dimer of Disperse Blue 56 or inhibition by a Disperse Blue 56 aggregate, but the latter appears much more likely. We present a detailed analysis of the system to provide further information that may help in the recognition of inhibition through aggregation.  相似文献   

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