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H Kacser

Publications and source records attributed to H Kacser.

At least 19 recordsLinked to original sources
PubMed1992

A strategy for increasing an in vivo flux by genetic manipulations. The tryptophan system of yeast.

Decreases in enzyme activity often have little effect on the flux carried by the pathway. Similarly, up-modulation of single genes, and hence of the dependent enzyme concentrations, is frequently found to be ineffective in increasing the flux in the pathway in which the enzyme occurs. This insensitivity to enzyme variation is demonstrated experimentally for five separate enzymes in the tryptophan synthesis system of yeast, first by down-modulation of the gene dose and secondly by increasing the dose using multi-copy vectors. Such a lack of response is discussed in terms of the concepts of metabolic control analysis. When these five enzymes, however, were simultaneously increased by a multi-copy vector carrying all five genes, a substantial elevation of the flux to tryptophan was observed. These findings revealed a new phenomenon, namely the more than additive effects on the flux of simultaneous elevations of several enzyme activities.

Down-Regulation↗
PubMed1990

Enzyme kinetics and metabolic control. A method to test and quantify the effect of enzymic properties on metabolic variables.

It is usual to study the sensitivity of metabolic variables to small (infinitesimal) changes in the magnitudes of individual parameters such as an enzyme concentration. Here, the effect that a simultaneous change in all the enzyme concentrations by the same factor alpha (Co-ordinate-Control Operation, CCO) has on the variables of time-dependent metabolic systems is investigated. This factor alpha can have any arbitrary large value. First, we assume, for each enzyme measured in isolation, the validity of the steady-state approximation and the proportionality between reaction rate and enzyme concentration. Under these assumptions, any time-invariant variable may behave like a metabolite concentration, i.e. S alpha = Sr (S-type), or like a flux, i.e. J alpha = alpha Jr (J-type). The subscripts r and alpha correspond to the values of the variable before and after the CCO respectively. Similarly, time-dependent variables may behave according to S alpha (t/alpha) = Sr (t) (S-type) or to J alpha (t/alpha) = alpha J r (t) (J-type). A method is given to test these relationships in experimental systems, and to quantify deviations from the predicted behaviour. A positive test for deviations proves the violation of some of the assumptions made. However, the breakdown of the assumptions in an enzyme-catalysed reaction, studied in isolation, may or may not affect significantly the behaviour of the system when the component reaction is embedded in the metabolic network.

Analysis of Variance↗
PubMed1990

Enzyme-enzyme interactions and control analysis. 1. The case of non-additivity: monomer-oligomer associations.

Two usual assumptions of the treatment of metabolism are: (a) the rates of isolated enzyme reactions are additive, i.e., that rate is proportional to enzyme concentration; (b) in a system, the rates of individual enzyme reactions are not influenced by interactions with other enzymes, i.e. that they are acting independently, except by being coupled through shared metabolites. On this basis, control analysis has established theorems and experimental methods for studying the distribution of control. These assumptions are not universally true and it is shown that the theorems can be modified to take account of such deviations. This is achieved by defining additional elasticity coefficients, designated by the symbol pi, which quantify the effects of homologous and heterologous enzyme interactions. Here we show that for the case of non-proportionality of rate with enzyme concentration, (pi ii not equal to 1), the summation theorems are given by (Formula: see text). The example of monomer-oligomer equilibria is used to illustrate non-additive behaviour and experimental methods for their study are suggested.

Catalysis↗
PubMed1990

Enzyme-enzyme interactions and control analysis. 2. The case of non-independence: heterologous associations.

The association of different enzymes into a complex may induce changes in the kinetic parameters of its component enzymes. This implies that they cannot be treated as independent catalysts. It will affect the formulations and theorems of control analysis and necessitates the introduction of additional elasticities reflecting the effect of one enzyme on the rate of another. We show how this is achieved as an extension of the classical treatment. We present modified summation and connectivity theorems incorporating both homologous and heterologous interactions. The case of channelling of metabolites in such complexes is considered and an experimental method for its detection is suggested.

Catalysis↗
PubMed1990

Control analysis of transition times in metabolic systems.

The transition time, tau, of a metabolic system is defined as the ratio of the metabolite concentrations in the system, sigma, to the steady-state flux, J. Its value reflects a temporal characteristic of the system as it relaxes towards the steady state. Like other systemic properties, the value of tau will be a function of the enzyme activities in the system. The influence of a particular enzyme activity on tau can be quantified by a Control Coefficient, C tau ei. We show that it is possible to derive a Summation Theorem sigma ni = 1 C tau ei = -1 and a Connectivity Theorem sigma ni = 1 C tau ei.epsilon viSk = -Sk/sigma. We establish a 'sign rule' that predicts the order of positive and negative Control Coefficients in a sequence.

Enzymes↗
PubMed1989

Control analysis of time-dependent metabolic systems.

Metabolic Control Analysis is extended to time dependent systems. It is assumed that the time derivative of the metabolite concentrations can be written as a linear combination of rate laws, each one of first order with respect to the corresponding enzyme concentration. The definitions of the control and elasticity coefficients are extended, and a new type of coefficient ("time coefficient", "T") is defined. First, we prove that simultaneous changes in all enzyme concentrations by the same arbitrary factor, is equivalent to a change in the time scale. When infinitesimal changes are considered, these arguments lead to the derivation of general summation theorems that link control and time coefficients. The comparison of two systems with identical rates, that only differ in one metabolite concentration, leads to a method for the construction of general connectivity theorems, that relate control and elasticity coefficients. A mathematical proof in matrix form, of the summation and connectivity relationships, for time dependent systems is given. Those relationships allow one to express the control coefficients in terms of the elasticity and time coefficients for the case of unbranched pathway.

Elasticity↗
PubMed1987

Dominance, pleiotropy and metabolic structure.

It is a common observation that most mutants have similar dominance relations for all the characters they are known to affect. As a model of pleiotropic effects we investigate a branched pathway where the two outputs represent two characters whose variation is affected by changes in any of the genetically specified enzymes in the system. We consider the effects on the phenotype (fluxes or intermediate metabolites) of substitutions at one locus represented by enzyme activities of the two homozygotes (mutant and wild type) and that of the heterozygote. Dominance indices for the characters pleiotropically connected by the metabolic system are calculated. We show that if enzymes behave 'linearly,' (first order), that is if saturation and feedback inhibition or other nonlinearities are absent, all fluxes and pools have identical dominance relations. The presence of such nonlinearity, however, leads to differences in dominance between different characters and we define the conditions where such differences can be important.

Enzymes↗
PubMed1986

Metabolic control analysis of moiety-conserved cycles.

Moiety-conserved cycles are metabolic structures that interconvert different forms of a chemical moiety (such as ATP-ADP-AMP, the different forms of adenylate), while the sum of these forms remains constant. Their metabolic behaviour is treated within the framework of control analysis [Kacser, H. & Burns, J.A. (1973) Symp. Soc. Exp. Biol 27, 65-104]. To explain the importance of the conserved sum of cycle metabolites as a parameter of the system, the cycle is first regarded as a 'black box'. The interactions of the cycle with the rest of the system are expressed in terms of 'cycle elasticities' and 'cycle control coefficients' by the usual connectivity properties. The conserved sum is seen to be an 'external' parameter in the sense that its effect is described by a combined response expression. All cycle coefficients can be written in terms of elasticities and concentrations of cycle metabolites. The treatment shows how connectivity expressions should be modified when moiety-conserved cycles are present and establishes new summation and connectivity properties. The analysis is applied to a two-member moiety-conserved cycle and its general application is discussed.

Catalysis↗
PubMed1986

Kinetics of metabolic pathways. A system in vitro to study the control of flux.

A method for determining Control Coefficients is proposed for systems studied in vitro and applied to a model pathway. Rat liver extract, which converts glucose into glycerol 3-phosphate, was used with the addition to the incubation mixture of fructose-bisphosphate aldolase, triose-phosphate isomerase and glycerol-3-phosphate dehydrogenase as 'auxiliary' enzymes, which leaves all the control on the first three enzymes. The flux of the metabolic pathway was recorded by assaying NADH decay. Flux Control Coefficients (CJE) of hexokinase, glucose-6-phosphate isomerase and phosphofructokinase were calculated by titration of the system with increasing quantities of extraneous enzymes. It is shown that the summation property is fulfilled. The applicability of this procedure to study the control in any metabolic pathway is discussed. Possible relevance of the method to conditions in vivo and its limitations are considered.

Animals↗
PubMed1985

Depression of enzyme synthesis in response to arginine limitation in Neurospora crassa.

Ornithine carbamoyltransferase and argininosuccinase, two enzymes involved in arginine synthesis, are regulated by cross-pathway amino acid control in Neurospora and show derepression in response to limitation of any one of a number of amino acids. The effects of varying the severity of arginine limitation upon the synthesis of these enzymes, in mycelial cultures of an arginine auxotrophic strain, are reported here. Depression occurred at arginine concentrations sufficient to allow normal rates of protein accumulation, leading to increases of not more than fourfold in the absolute rate of enzyme synthesis. On the other hand, differential rates of enzyme synthesis increased progressively up to 20-fold or more under extreme conditions of arginine limitation that also limit net protein synthesis. The major part of the derepression response thus occurred at arginine concentrations that allowed low net rates of protein synthesis. The physiological significance of this is not yet understood. Our evidence suggests that these responses were mediated entirely through the cross-pathway control system, and may not be untypical (allowing for variations in magnitude) of depression resulting through this mechanism in Neurospora.

Amino Acids↗
PubMed1978

Genetic variation in activity of the enzymes of glycolysis and gluconeogenesis between inbred strains of mice.

Variation in the activity of 21 liver and 15 erythrocyte enzymes between seven inbred strains of mice has been studied in a single area of metabolism, glycolysis and gluconeogenesis. Most of the variation between the strains is genetic. From the variation within and between inbred strains heritabilities (H2) were determined. Out of 35, 26 showed significant values above 0.4. A comparison with previously published work suggests that enzyme activities have mainly dominance and interaction components of variance, and this is discussed in relation to the variation in quantitative characters such as growth. In nine of the pairwise comparisons of the strains, the activity of the enzyme varied more than two-fold. In these cases the genetics and biochemistry of the enzyme was studied; F2 progeny were produced and assessed for segregation, and the heat stability of the enzyme was determined. No unequivocal segregation was observed, although in one case we found a considerable difference in heat stability. The variations found were not considered to be great enough to be useful as models of human inborn errors of metabolism or to study metabolic control. If such variants are to be found, sources of variation other than inbred strains must be used.

Animals↗