Biomedical subjects
Publications and source records attributed to R G Duggleby.
Omeprazole inhibited two thiamin diphosphate-dependent enzymes, pyruvate decarboxylase (EC 4.1.1.1, PDC) from Zymomonas mobilis and transketolase (EC 2.2.1.1, TK) from human erythrocytes. Inhibition of PDC was competitive with the coenzyme with a Ki value of 42 +/- 3 microM, whereas inhibition of TK was complex.
A tryptophan residue at position 487 in Zymomonas mobilis pyruvate decarboxylase was altered to leucine by site-directed mutagenesis. This modified Z. mobilis pyruvate decarboxylase was active when expressed in Escherichia coli and had unchanged kinetics towards pyruvate. The enzyme showed a decreased affinity for the cofactors with the half-saturating concentrations increasing from 0.64 to 9.0 microM for thiamin diphosphate and from 4.21 to 45 microM for Mg2+. Unlike the wild-type enzyme, there was little quenching of tryptophan fluorescence upon adding cofactors to this modified form. The data suggest that tryptophan-487 is close to the cofactor binding site but is not required absolutely for pyruvate decarboxylase activity. Substitution of asparagine, threonine or glycine for aspartate-440, a residue which is conserved between many thiamin diphosphate-dependent enzymes, completely abolishes enzyme activity.
A recent article (Leonhardt et al. Comput. Methods Prog. Biomed. 32 (1990) 345-350) described the analysis of biological turnover data using a model involving an exponential decay from an initial value to a plateau. The authors included a BASIC program for this analysis, claiming that no comparable programs have been published. In fact, there are many programs available which can be used for this purpose and several are greatly superior to that proposed.
When the progress curve for an enzyme catalysed reaction follows the integrated Michaelis-Menten equation, the maximum velocity and Michaelis constant can be determined from a single such curve. In this paper, an experimental design for collecting the data is proposed which is close to optimum in the sense that it produces the smallest standard error in the estimate of the Michaelis constant. This design involves choosing an initial substrate concentration which is approx. two or three times the value of the Michaelis constant.
A new type of enzyme kinetic mechanism is suggested by which catalysis may be viewed as a chain reaction. A simple type of one-substrate/one-product reaction mechanism has been analysed from this point of view, and the kinetics, in both the transient and the steady-state phases, has been reconsidered. This analysis, as well as literature data and theoretical considerations, shows that the proposed model is a generalization of the classical ones. As a consequence of the suggested mechanism, the expressions, and in some cases even the significance of classical constants (Km and Vmax.), are altered. Moreover, this mechanism suggests that, between two successive enzyme-binding steps, more than one catalytic act could be accomplished. The reaction catalysed by alcohol dehydrogenase was analysed, and it was shown that this chain-reaction mechanism has a real contribution to the catalytic process, which could become exclusive under particular conditions. Similarly, the mechanism of glycogen phosphorylase is considered, and two partly modified versions of the classical mechanism are proposed. They account for both the existing experimental facts and suggest the possibility of chain-reaction pathways for any polymerase.
To study the mechanism of re-activation of Zymomonas mobilis pyruvate decarboxylase apoenzyme by its cofactors thiamin diphosphate and Mg2+, cofactor-free enzyme was prepared by dialysis against 1 mM-dipicolinic acid at pH 8.2. This apoenzyme was then used in a series of experiments that included determination of: (a) the affinity towards one cofactor when the other was present at saturating concentrations; (b) cofactor-binding rates by measuring the quenching of tryptophan fluorescence on the apoenzyme; (c) the effect of replacement of cofactors with various analogues; (d) the stoichiometry of bound cofactors in holoenzyme; and (e) the molecular mass of apoenzyme by gel filtration. The results of these experiments form the basis for a proposed model for the re-activation of Z. mobilis pyruvate decarboxylase apoenzyme by its cofactors. In this model there exists two alterative but equivalent pathways for cofactor binding. In each pathway the first step is an independent reversible binding of either thiamin diphosphate (Kd 187 microM) or Mg2+ (Kd 1.31 mM) to free apoenzyme. When both cofactors are present, the second cofactor-binding step to form active holoenzyme is a slow quasi-irreversible step. This second binding step is a co-operative process for both thiamin diphosphate (Kd 0.353 microM) and Mg2+ (Kd 2.47 microM). Both the apo- and the holo-enzyme have a tetrameric subunit structure, with cofactors binding in a 1:1 ratio with each subunit.
Measuring the initial velocity is difficult in some enzyme assays where a significant fraction of the substrate is consumed. Here a solution to this problem is proposed; the time to produce a fixed amount of reaction product is measured. This time is inversely proportional to the initial velocity, and is related to the maximum velocity and Michaelis constant by a simple equation and linear plot. The method is illustrated using the reaction catalysed by pyruvate kinase.
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We have shown by appropriate modification of the translational signals and using the strong T7 RNA polymerase promoter phi 10, that a cloned Saccharomyces cerevisiae pyruvate decarboxylase gene (pdc1) can be expressed in Escherichia coli. This protein, which migrated as a single band on SDS-polyacrylamide gels, was found to have a subunit molecular mass of approximately 62 kDa, similar to that of the enzyme produced by yeast. Polyclonal antibodies raised against purified yeast pyruvate decarboxylase recognized this bacterially produced protein. We found that this recombinant enzyme is active, indicating that the homotetramer encoded by the pdc1 gene is functional.
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Often the Michaelis constant of an enzyme will be determined several times. This may be done for various reasons such as ensuring reproducibility, comparing different enzyme preparations, or examining the effects of variations in experimental conditions. In these circumstances, two questions arise. First, how can the various estimates of the Michaelis constant be compared to determine whether they are the same within the limits of experimental variation? Secondly, if they are all the same, how can the values be combined to give an overall estimate? These questions are addressed here and a solution proposed in which the sets of data are pooled and analyzed with a separate maximum velocity for each set but a common Michaelis constant. The pooled data are partitioned in suitable ways and reanalyzed to examine, by means of a variance ratio test, whether a single Michaelis constant gives a satisfactory fit to the data.
Pyruvate decarboxylase from Zymomonas mobilis is inhibited by 3-hydroxypyruvate, which can also act as a poor substrate. While catalysing the decarboxylation of this alternative substrate, the enzyme undergoes a progressive but partial inactivation over several hours. The extent of inactivation depends upon the pH and upon the concentration of 3-hydroxypyruvate. After partial inactivation and removal of unchanged 3-hydroxypyruvate, enzymic activity recovers slowly. We suggest that inactivation results from accumulation of enzyme-bound glycollaldehyde, which is relatively stable, possibly because it is dehydrated to form an acetyl group.
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.
Concentration gradients are often used in separations based on chromatographic, electrophoretic and centrifugal methods. In this report, a BASIC computer program for calculating and graphically representing gradients is described. This GRADIENT program is intended to be run on IBM-compatible computers.
The computer analysis of progress curves for enzyme-catalysed reactions involves a series of mathematical and computational tasks. The three most daunting of these are the derivation of an integrated rate equation, solving this equation so that the amount of product formed by the reaction at any time can be calculated, and incorporating this solution into a non-linear-regression computer program. This paper describes the basis of a computer program that greatly simplifies the problem. The proposed mechanism is specified in the familiar kinetic constant form, which is automatically translated into a program capable of fitting this mechanism to a series of experimental progress curves. The approach is illustrated for a reversible reaction with one substrate and one product, and tested with some data obtained for the fumarase reaction. A copy of the program has been deposited as Supplementary Publication SUP 50148 (13 pages) at the British Library Document Supply Centre, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1989) 257, 5.
The spectrum of a chromophore may change as a result of perturbations in its environment. The spectral changes resulting from the perturbation are often followed by measurements at just one or two wavelengths but it is usually no more difficult to collect entire spectra. The problem comes in analysing the data from such a series of spectra. In this paper we will suggest a simple procedure in which the spectrum observed under any particular set of conditions may be considered to consist of the sum of two distinct spectral forms. The method, which is free of any assumptions regarding the quantitative relationship between the perturbation and the extent of spectral change, defines any given spectrum in terms of an apparent molar fraction of the contributing spectral forms. The variation of this apparent molar fraction provides information from which a quantitative relationship can be developed to describe the dependence of the spectral change on the perturbant. The method is illustrated using the model system of phenol red protonation and is applied to the characterization of the binding of azide ions to cobalt-substituted carbonic anhydrase.
Many organophosphorus compounds are irreversible inhibitors of acetylcholinesterase. The methods used in the literature to determine the inhibition kinetic constants usually involve either manual determination of the slope at various points along the inhibition progress curve or fitting polynomials to the curve. The present study investigates the use of non-linear-regression analysis to determine the various parameters. A method is suggested that yields accurate values for the inhibition constants under a range of circumstances.
Current methods for fitting integrated rate equations to enzyme progress curves treat each observation as if it were an independent measurement. When the data are obtained by taking several successive readings from each of a series of progress curves, the data will not be truly independent and will exhibit autocorrelation. Here we propose a simple pragmatic extension of integrated rate equations which takes account of first-order autocorrelations. The value of the method is assessed when applied to five sets of experimental data.