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PubMed1988

Theory formation in postulating enzyme kinetic mechanisms: reasoning with constraints.

This paper reports on a prototype system for modeling and analyzing the expert reasoning involved in postulating enzyme kinetic mechanisms. It involves data-driven, theory-driven, and analogical components of reasoning within a generate-and-test cycle. Its central component is a set of domain-specific "filters" for matching experimentally and theoretically derived constraints. The input to the system consists of an abstracted qualitative description of an experiment and prior knowledge reported in the literature. Its output shows how the results match those expected for a set of postulated reaction mechanism models and also provides a trace of which features do or do not match each of the candidate topological models. Results, constraints, and models are all analyzed and compared to those from other, similar experiments. We deduced rules for interpreting the qualitative features of enzyme kinetic experiments from natural language descriptions in the literature and verified that the rules were correct by predicting the results for typical mechanisms. We obtained the correct behavior for all 37 states of a complex enzyme mechanism involving three substrates and three products. We tested our system on data from several published reports dealing with the enzyme hexokinase and obtained detailed listings of the differences in conclusions and interpretation reported in several journal articles. This system, which provides qualitative representations of enzyme kinetic results, should facilitate further experimentation on theory formation in enzyme kinetics and lead to more efficient experimental designs.

Artificial Intelligence↗
PubMed2005

A generalized numerical approach to rapid-equilibrium enzyme kinetics: application to 17beta-HSD.

A generalized numerical treatment of rapid-equilibrium enzyme kinetics is presented. This new approach relies on automatic computer derivation of the underlying mathematical model (a system of simultaneous nonlinear algebraic equations) from a symbolic representation of the reaction mechanism (a system of biochemical equations) provided by the researcher. The method allows experimental biochemists to analyze initial-rate enzyme kinetic data without having to use any mathematical equations. An illustrative example is based on the inhibition kinetics of 17beta-hydroxysteroid dehydrogenase type 5 by a class of natural compounds. A computer implementation of the new method, a newly modified software package DYNAFIT [Kuzmic, P., 1996. Program DYNAFIT for the analysis of enzyme kinetic data: application to HIV proteinase. Anal. Biochem. 237, 260-273], is freely available to all academic researchers.

17-Hydroxysteroid Dehydrogenases↗
PubMed1986

Application of progress curve analysis to in situ enzyme kinetics using 1H NMR spectroscopy.

The steady-state kinetics of enzymes in tissues, cells, and concentrated lysates can be characterized using high-resolution nuclear magnetic resonance spectroscopy; this is possible because almost invariably there are differences in the spectra of substrates and products of a reaction and these spectra are obtainable even from optically opaque samples. We used 1H spin-echo NMR spectroscopy to study the hydrolysis of alpha-L-glutamyl-L-alanine by cytosolic peptidases of lysed human erythrocytes. Nonlinear regression of the integrated Michaelis-Menten expression onto the progress-curve data yielded, directly, estimates of Vmax and Km for the hydrolase; a procedure for analyzing progress curves in this manner was adapted and compared with a commonly used procedure which employs the Newton-Raphson algorithm. We also performed a sensitivity analysis of the integrated Michaelis-Menten expression; this yielded equations that indicate under what conditions estimates of Km and Vmax are most sensitive to variations in experimental observables. Specifically, we showed that the most accurate estimates of the steady-state parameters from analysis of progress curves are obtained when the initial substrate concentration is much greater than Km. Furthermore, estimates of these parameters obtained by such an analysis are most sensitive to data obtained when the reaction is 60-80% complete, having started with the highest practicable initial substrate concentration.

Dipeptides↗
PubMed1999

Multi-enzyme kinetic analysis of glycolipid biosynthesis.

Gangliosides are acidic glycosphingolipids synthesized sequentially by a series of glycosyltransferases acting in parallel biosynthetic pathways. While most glycosyltransferases are highly specific, some, however, may catalyze equivalent steps in each pathway using different gangliosides as substrates (e.g. N-acetylgalactosaminyltransferase, sialyltransferase-IV). A multi-enzyme kinetic analysis was developed on the condition that serial enzymatic reactions operate below substrate saturation. A multi-enzyme kinetic analysis enabled a simultaneous calculation of the Vmax/Km value of each enzyme derived from the equilibrium concentration of the respective substrate. Substrate concentrations [S] were determined by radioactive labelling of gangliosides in intact cells with the precursor sugars [14C]galactose and [14C]glucosamine, followed by high-performance thin-layer chromatography and autoradiography of the radiolabelled glycolipids. On the basis of Michaelis-Menten kinetics, Vmax/Km values were derived from [S] by a system of linear equations. The procedure was used to analyze the development of the glycolipid composition during differentiation of rat gliomaxmurine neuroblastoma (NG108-15) cells. The Vmax/Km values calculated by multi-enzyme kinetic analysis were consistent with the kinetic data obtained with solubilized enzymes. Application of multi-enzyme kinetic analysis to published data on the correlation of enzyme activities with ganglioside levels in various cell lines and tissues indicated the validity of this method for analysis of the glycolipid biosynthesis, in particular, of its initial steps. On the basis of the kinetic analysis, it is suggested that the cell lines can be divided into two groups with respect to the substrate pools of GM3 used by sialyltransferase-II and N-acetylgalactosaminyltransferase-I. The first group encompasses the majority of the neuroblastoma cell lines and the embryonic rat brain where the two enzymes share a common pool of GM3. In the second group, the two enzymes do not compete for the same pool of GM3, indicating a different subcellular localization of CMP-NeuAc:GM3 alpha2-8-sialyltransferase and UDP-N-acetylgalactosaminyl:GM3 N-acetylgalactosaminyltransferase. In this study, the theory of a multi-enzyme kinetic analysis is discussed and its application to analysis of the glycolipid biosynthesis in neuroblastoma cells is demonstrated. A multi-enzyme kinetic analysis can be applied to other biosynthetic pathways and provides the advantage of analyzing kinetic data with intact cells or tissue samples.

Animals↗
PubMed1990

Automatic computation of enzyme kinetics by HPLC.

Enzyme activity can be easily measured by HPLC using traces of the product itself as an internal standard. Our procedure involved the development of an equation using the experimental data obtained in the kinetic assay. The entire procedure can thus be automated and a computer program is presented here for facilitating the assay and saving time. The determination of the activity of NAD kinase is reported as an example.

Algorithms↗
PubMed1990

Applications of graph theory to enzyme kinetics and protein folding kinetics. Steady and non-steady-state systems.

Graphic methods have proved to be very useful in enzyme kinetics, as reflected in both raising the efficiency of performing calculations and aiding in the analysis of catalytic mechanisms. The kinetic relations among protein folding states are very similar to those between enzyme-catalyzed species. Therefore, it should be equally useful to provide a visually intuitive relation between kinetic calculations and folding mechanisms for protein folding kinetics, as manifested by the graphic rules in enzyme kinetics. It can actually be anticipated that, due to increasing interest in protein folding, the graphic method will become an important tool in folding kinetics as well. Based on the recent progress made in graphic methods of enzyme kinetics, in this review four graphic rules are summarized, which can be used to deal with protein folding systems as well as enzyme-catalyzed systems. Rules 1-3 are established for deriving the kinetic equations for steady-state processes and Rule 4 for those in the case of non-steady-state processes. In comparison with conventional graphic methods, which can only be applied to a steady-state system, the current rules have the following advantages: (1) Complicated and tedious calculations can be greatly simplified. (2) A lot of wasted labor can be turned away. (3) Final results can be double-checked by a formula provided in each of the graphic rules. (4) Transient kinetic systems can also be treated. The mathematical proof of Rules 1-4 is given in appendices A-D, respectively.

Enzymes↗
PubMed2000

Microsomal binding of amitriptyline: effect on estimation of enzyme kinetic parameters in vitro.

The effect of binding of amitriptyline to human liver microsomes and to microsomes from human B-lymphoblastoid cells on the estimation of enzyme kinetic parameters describing N-demethylation to nortriptyline was investigated using a combination of microsomal binding and in vitro enzyme kinetic studies. Quantitative binding in both matrices increased with higher microsomal protein concentrations (free fractions 0.88-0.32 at 100-500 microg protein/ml in human liver microsomes and 0.82-0.26 at 250-1000 microg protein/ml in microsomes from B-lymphoblastoid cells) and was independent of amitriptyline concentration over a concentration range of 0.2 to 200 microM. Addition of heat-inactivated microsomal protein (50-450 microg/ml) to native human liver microsomes (50 microg/ml) reduced the amitriptyline N-demethylation rate in a protein concentration dependent manner. This effect was greater at lower substrate concentrations and was overcome by saturating concentrations of substrate, thereby decreasing the apparent affinities of the high- and low-affinity components of the N-demethylation process, with minimal effect on the net V(max). Addition of metabolically inactive microsomes from untransfected human lymphoblastoid cells (750 microg/ml) to CYP2C19 (250 microg/ml protein) increased the apparent K(m) value for amitriptyline N-demethylation by 3.5-fold and increased the uncompetitive substrate inhibition constant (K(s)) by 2.2-fold, making substrate inhibition essentially undetectable. A similar effect was seen with CYP3A4, with a 1.8-fold increase in the S(50) (substrate concentration at which half-maximal velocity of a Hill enzyme is achieved). Microsomal binding did not alter the V(max) of either CYP isoform to any appreciable extent. These findings emphasize the importance of incorporating microsomal binding in the estimation of enzyme kinetic parameters in vitro and making appropriate corrections for unbound drug concentrations.

Algorithms↗
PubMed1980

Determination of urinary beta-glucuronidase activity. Single-point versus enzyme kinetic measuring system.

beta-Glucuronidase activity was determined in dialyzed and undialyzed urine from 50 healthy adult males by single-point and enzyme kinetic measuring systems. The enzyme had a pH optimum of 5.2 and a Michaelis constant of 1.465 +/- 0.206 mmol/l (mean +/- SD) of phenolphthalein glucuronide. Substrate inhibition occurred at a concentration of 0.006 mol/l in one fourth of the urine samples. A significant amount of a competitive inhibitor, D-glucaro-1,4-lactone, was present in one third of the specimens. The activity measured by the single-point determination was always lower than the maximal velocity, particularly in the presence of the competitive inhibitor and substrate inhibition. Such pitfalls could be avoided by the enzyme kinetic method which not only permits the detection of substrate inhibition of the enzyme and the elimination of 2-hour dialysis of urine, but also allows the determination of both the maximal velocity of the enzyme and the quantity of D-glucaro-1,4-lactone in urine.

Glucaric Acid↗
PubMed1981

Fitting of enzyme kinetic data without prior knowledge of weights.

A method is described for fitting equations to enzyme kinetic data that requires minimal assumptions about the error structure of the data. The dependence of the variances on the velocities is not assumed, but is deduced from internal evidence in the data. The effect of very bad observations ('outliers') is mitigated by decreasing the weight of observations that give large deviations from the fitted equation. The method works well in a wide range of circumstances when applied to the Michaelis-Menten equation, but it is not limited to this equation. It can be applied to most of the equations in common use for the analysis of steady-state enzyme kinetics. It has been implemented as a computer program that can fit a wide variety of equations with two, three or four parameters and two or three variables.

Catalysis↗
PubMed2004

A novel thermodynamic relationship based on Kramers Theory for studying enzyme kinetics under high viscosity.

In most studies of enzyme kinetics it has been found sufficient to use the classical Transition State Theory (TST) of Eyring and others. This theory was based on the solvent being an ideal dilute substance treated as a heat bath. However, enzymes found in organisms adapted to very low (psychrophiles) and very high (thermophiles) temperatures are also subjected to variable solute concentrations and viscosities. Therefore, the TST may not always be applicable to enzyme reactions carried out in various solvents with viscosities ranging from moderate to very high. There have been numerous advances in the theory of chemical reactions in realistic non-ideal solvents such as Kramers Theory. In this paper we wish to propose a modified thermodynamic equation, which have contributions from kcat, Km and the viscosity of the medium in which the enzyme reaction is occurring. These could be very useful for determining the thermodynamics of enzymes catalyzing reactions at temperature extremes in the presence of substrate solutions of different compositions and viscosities.

Catalysis↗
PubMedDate not supplied

Steady state enzyme kinetics: experimental design and data analysis by microcomputer.

One of the most time-consuming, yet essential, operations involved in the steady state kinetic study of enzymes is the design and optimization of experimental conditions. A computer program was developed for the Sinclair ZX-81 (or TS-1000, 1500) microcomputer which will optimize substrate concentration for preliminary and subsequently more refined kinetic analysis of one, two or three substrate systems. This program also analyzes the data collected from these studies by linear regression, weighted linear regression or weighted non-linear regression. In addition to the above program several of the enzyme kinetic statistical analysis programs of Cleland (1979) have been translated from FORTRAN into BASIC and implemented on the ZX-81 and the TRS-80 model II. Inexpensive commercially available software was used to overcome the inability of the ZX-81 to read data files from magnetic tape making the data analysis programs easier to use.

Computers↗
PubMed2005

Experimental design for optimal parameter estimation of an enzyme kinetic process based on the analysis of the Fisher information matrix.

The investigation of enzyme kinetics is increasingly important, especially for finding active substances and understanding intracellular behaviors. Therefore, the determination of an enzyme's kinetic parameters is crucial. For this a systematic experimental design procedure is necessary to avoid wasting time and resources. The parameter estimation error of a Michaelis-Menten enzyme kinetic process is analysed analytically to reduce the search area as well as numerically to specify the optimum for parameter estimation. From analytical analysis of the Fisher information matrix the fact is obtained, that an enzyme feed will not improve the estimation process, but substrate feeding is favorable with small volume flow. Unconstrained and constrained process conditions are considered. If substrate fed-batch process design is used instead of pure batch experiments the improvements of the Cramer-Rao lower bound of the variance of parameter estimation error reduces to 82% for mu(max) and to 60% for K(m) of the batch values in average.

Animals↗
PubMed2000

Metabolism of ezlopitant, a nonpeptidic substance P receptor antagonist, in liver microsomes: enzyme kinetics, cytochrome P450 isoform identity, and in vitro-in vivo correlation.

The enzyme kinetics of the metabolism of ezlopitant in liver microsomes from various species have been determined. The rank order of the species with regard to the in vitro intrinsic clearance of ezlopitant was monkey >> guinea pig > rat >> dog > human. CJ-12,764, a benzyl alcohol analog, was observed as a major metabolite, and a dehydrogenated metabolite (CJ-12,458) was equally important in human liver microsomes. Scale-up of the liver microsomal intrinsic clearance data and correcting for both serum protein binding and nonspecific microsomal binding yielded predicted hepatic clearance values that showed a good correlation with in vivo systemic blood clearance values. Including microsomal binding was necessary to achieve agreement between hepatic clearance values predicted from in vitro data and systemic clearance values measured in vivo. Cytochrome P450 (CYP) 3A4, 3A5, and 2D6 demonstrated the ability to metabolize ezlopitant to CJ-12,458 and CJ-12,764. However, in liver microsomes, the CYP3A isoforms appear to play a substantially more important role in the metabolism of ezlopitant than CYP2D6, as assessed through the use of CYP-specific inhibitors, correlation to isoform-specific marker substrate activities, and appropriate scale-up of enzyme kinetic data generated in microsomes containing individual heterologously expressed recombinant CYP isoforms. The apparent predominance of CYP3A over CYP2D6 is consistent with observations of the pharmacokinetics of ezlopitant in humans in vivo.

Animals↗