Assessing risks of genetic engineering.
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Commerce↗
Biomedical subjects
J Kinderlerer
Publications and source records attributed to J Kinderlerer.
Exponential model for a regulatory enzyme. Computer program for the determination of the model constants from initial velocity data.
The paper describes a program (DESCENT) which evaluates the constants of the exponential model for a regulatory enzyme with up to four substrates or up to three substrates and one effector. The program operates by repetitive adjustment of the model constants so as to secure a better fit between the observed and calculated initial velocities of reaction. At each adjustment, all the constants are incremented simultaneously by amounts determined by a steepest descent criterion. The program is tested by artificial data with and without added error and it is shown that good estimates of the constants can be recovered.
Allosteric Regulation↗
The regulatory properties of yeast pyruvate kinase. Effect of fructose 1,6-bisphosphate.
The kinetics of pyruvate kinase from Saccharomyces cerevisiae were studied in assays at pH 6.2 at 25 degrees C as a function of the concentrations of the substrates ADP, phosphoenolpyruvate and Mg2+ and the concentration of the effector fructose 1,6-bisphosphate. The enzyme was activated by 100 mM-K+ and 32 mM-NH4+ throughout. It was found that an increase in the fructose bisphosphate concentration from 24 microM to 1.2 mM brings about a transition from a sigmoidal to a non-inflected form in the relationships v = f([phosphoenolpyruvate]) and v = f([Mg2+]) together with a large increase in the affinity of these substrates for the enzyme. The binding behaviour of ADP is barely affected by the same change in effector concentration. By contrast, increase in fructose bisphosphate concentration below 24 microM increases the affinity of the enzyme for all its substrates and the sigmoidicity of the corresponding velocity-substrate-concentration relationships. As a result of this change in behaviour it has been found impossible to represent all the data by the exponential model for a regulatory enzyme, and it is suggested (supported by comparisons with previous work) that the failure may reflect a secondary action of the effector upon the enzyme.
Adenosine Diphosphate↗
The regulatory properties of yeast pyruvate kinase. Effect of pH.
The kinetics of pyruvate kinase from Saccharomyces cerevisiae were studied at 25 degrees C as a function of the concentrations of the substrates ADP, phosphoenolpyruvate and Mg2+ and the effector H+ in the pH range 5-6.6. The enzyme was activated by 100 mM-K+ and 32 mM-NH4+ throughout. It was found that the data could be described by the exponential model for a regulatory enzyme. On that basis, it was concluded that the binding of H+ is positively interactive and that the protonated enzyme is catalytically inactive. It was also found that H+ interacts positively with phosphoenolpyruvate but negatively with both ADP and Mg2+.
Adenosine Diphosphate↗
The regulatory properties of yeast pyruvate kinase. Effects of NH4+ and K+ concentrations.
The kinetics of pyruvate kinase from Saccharomyces cerevisiae were studied at 25 degrees C and pH 6.2 as a function of the concentrations of ADP, phosphoenolpyruvate, Mg2+ and either NH4+ or K+. The data were analysed by the exponential model for four substrates, obtained by extension of the model described by Ainsworth, Kinderlerer & Gregory [(1983) Biochem. J. 209, 401-411]. On that basis, it was concluded that NH4+ binding is almost non-interactive but leads to the appearance of positive interaction in the velocity response to increase in its concentration because of positive interactions with phosphoenolpyruvate and Mg2+. The data obtained with K+ lead to the same conclusions and differ only in suggesting that NH4+ is bound more strongly to the enzyme than is K+. Both data sets are used as the basis for a discussion of the substrate interactions of pyruvate kinase and it appears therefrom that the heterotropic interactions accord with what is known of the events that take place at the active site during catalysis. The paper also reports a determination of the dissociation constants for the NH4+ complexes with ADP and phosphoenolpyruvate and an examination of the simultaneous activation of pyruvate kinase by K+ and NH4+ ions.
Adenosine Diphosphate↗
The regulatory properties of yeast pyruvate kinase.
The kinetics of pyruvate kinase from Saccharomyces cerevisiae were studied in assays at pH 6.2 where the relationships between the initial velocities of the catalysed reaction and the concentrations of the substrates ADP, phosphoenolpyruvate and Mg2+ are non-hyperbolic. The findings were represented empirically by the exponential model for a regulatory enzyme. The analysis shows that ADP, phosphoenolpyruvate and Mg2+ display positive homotropic interaction in their binding behaviour with (calculated) Hill slopes at half-saturation equal to 1.06, 2.35 and 3.11 respectively [Ainsworth (1977) J. Theor. Biol. 68, 391-413]. The direct heterotropic interaction between ADP and phosphoenolpyruvate is small and negative, but the overall interaction between these substrates becomes positive when their positive interactions with Mg2+ are taken into account. The heterotropic interactions of the substrates, though smaller in magnitude, are comparable with those revealed by the rabbit muscle enzyme [Ainsworth, Kinderlerer & Gregory (1983) Biochem. J. 209, 401-411], and it is suggested that they have a common origin in charge interactions within the active site.
Adenosine Diphosphate↗
The regulatory properties of rabbit muscle pyruvate kinase. The influence of substrate concentrations.
The kinetics of rabbit muscle pyruvate kinase were studied in assays at pH 7.4, where the relationships between the initial velocities of the catalysed reaction and the concentrations of substrates ADP, phosphoenolpyruvate and Mg2+ are non-hyperbolic. The data were used to test the applicability of the exponential model for a regulatory enzyme, which has been here extended to describe the behaviour of a three-substrate enzyme. It appears that the data can be represented by the model and as a result permit the conclusion that the substrates influence one another's binding by the same type of charge interactions that are evident in the Michaelis-Menten kinetics of the enzyme observed at pH 6.2. Evidence is also presented indicating that MgADP acts as a dead-end inhibitor of the enzyme at pH 7.4.
Adenosine Diphosphate↗
The regulatory properties of rabbit muscle pyruvate kinase. The influence of effector concentrations.
The initial velocity of the reaction catalysed by rabbit muscle pyruvate kinase was studied as a function of the concentrations of the modifiers phenylalanine and fructose 1,6-bisphosphate under conditions where the relationships between the initial velocities and the concentrations of substrates are non-hyperbolic. It is shown that these data can be represented by the exponential model for a regulatory enzyme.
Adenosine Diphosphate↗
The identification of unbranched enzyme kinetic mechanisms by computer analysis of qualitative information: logic of the method.
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Computers↗
Exponential model for a two-ligand, regulatory enzyme. Part 1: computer programs for the determination of the model constants from initial velocity data.
The exponential model for a regulatory enzyme with two ligands (either two substrates or one substrate and an effector) provides an expression for the initial velocity of the catalysed reaction in terms of the fractional saturation of the enzyme by each ligand. This paper describes a program which determines the constants of the model from velocity data, measured with respect to the concentrations of the two ligands, when the corresponding fractional saturations are unknown. Listings of the essential routines, written in BASIC, are provided.
Catalysis↗
Exponential model for a two-ligand, regulatory enzyme. Part 2: Performance tests of the 'INDEXP' computer program for the determination of model constants from initial velocity data. I. Artificial data.
The exponential model for a regulatory enzyme describes the relationship between the initial velocity of the catalysed reaction and the concentration of two ligands. A program, entitled 'INDEXP' has been designed to analyse rate data in terms of the model (Kinderlerer et at., 1981) and, in this report, its performance is examined when presented with artificial data generated from known constants and random, normally distributed error. It is shown that 'INDEXP' is able to recover good estimates of the constants.
Catalysis↗
Exponential model for a two-ligand, regulatory enzyme. Part 3: Performance tests of INDEXP computer programs for determination of model constants from initial velocity data. 2. Experimental data.
The exponential model for a regulatory enzyme describes the relationship between the initial velocity of the catalysed reaction and the concentration of two ligands. A program, entitled 'INDEXP' has been devised to analyse rate data in terms of the model (Kinderlerer et al., 1981) and, in this report, its performance is examined when presented with experimental initial velocity data taken from the literature. It is shown that the two-ligand exponential model can satisfactorily rationalise experimental data with five linked constants (Ainsworth and Gregory, 1978); as a result the influence of ligand concentrations on the catalysed reactions can be described in rather simple terms.
Animals↗
The effect of pH on enzyme-catalysed reactions: a computer program to determine pK values of enzyme ionisable groups from kinetic data.
The paper describes a non-linear multiple regression program to analyse the kinetic parameters Vm and Km as functions of pH, assuming that the activity of particular enzyme forms is determined by the ionisation of two groups, the active form of the enzyme being the half-ionised species.
Catalysis↗
Computer programs for use in enzyme kinetic studies.
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Computers↗
The exponential model for a regulatory enzyme: a computer program to determine the constants for the binding of a single ligand.
The paper describes a non-linear multiple regression program to analyse data arising from the binding of a single ligand by a protein under the assumption that the reaction can be represented by the exponential model for a regulatory enzyme.
Computers↗
A computer program to derive the rate equations of enzyme catalysed reactions with up to ten enzyme-containing intermediates in the reaction mechanism.
The paper describes a program, designed for a desk-top computer, which can be used to derive the rate equations of enzyme catalysed reactions with up to ten enzyme-containing intermediates included in the mechanism. The program allows the rate equation to be presented in simplified forms of practical use and in a variety of formats.
Catalysis↗
The derivation of second degree rate equations arising from two-substrate, two-product enzyme catalysed reactions whose catalytic cycle is branched.
A selection of two-substrate, two-product enzyme catalysed reactions with alternate catalytic cycles and second degree rate equations was examined by the computer program described by Kinderlerer and Ainsworth (1976). It is shown that the numerator terms of the rate equations are particularly simple, easy to derive and capable of creating a broad division of the mechanisms considered. THE NUMERATOR TERMS ALSO HAVE THE ADVANTAGE OF BEING UNAFFECTED BY DEAD END INHIBITION OR THE PRESENCE OF Theorell-Chance reactions. It is then shown that the relatively small group of mechanisms, isolated by the numerator analysis, can be distinguished from each other by considering the denominator terms of the rate equation that are second degree in the concentration of the varied substrate.
Catalysis↗