Electrostatics and diffusion of molecules in solution: simulations with the University of Houston Brownian dynamics program

This paper examines whether the in vivo behavior of yeast glycolysis can be understood in terms of the in vitro kinetic properties of the constituent enzymes. In nongrowing, anaerobic, compressed Saccharomyces cerevisiae the values of the kinetic parameters of most glycolytic enzymes were determined. For the other enzymes appropriate literature values were collected. By inserting these values into a kinetic model for glycolysis, fluxes and metabolites were calculated. Under the same conditions fluxes and metabolite levels were measured. In our first model, branch reactions were ignored. This model failed to reach the stable steady state that was observed in the experimental flux measurements. Introduction of branches towards trehalose, glycogen, glycerol and succinate did allow such a steady state. The predictions of this branched model were compared with the empirical behavior. Half of the enzymes matched their predicted flux in vivo within a factor of 2. For the other enzymes it was calculated what deviation between in vivo and in vitro kinetic characteristics could explain the discrepancy between in vitro rate and in vivo flux.

Can yeast glycolysis be understood in terms of in vitro kinetics of the constituent enzymes? Testing biochemistry.

Can yeast glycolysis be understood in terms of in vitro kinetics of the constituent enzymes

The theoretical background is presented for (a) the relaxation towards equilibrium of drug-induced membrane currents, and (b) the fluctuations of membrane current about its equilibrium value that originate in the opening and closing of membrane ion channels. General expressions are given that relate the relaxation current, autocovariance function, spectral density function, fluctuation variance and mean open channel lifetime to the rate constants and single channel conductances for any theory of drug action based on the law of mass action. The question of how much can be validly inferred from experimental spectra that appear to have only one component is discussed. The equations are illustrated by their application to some simple theories of drug action that are currently under consideration.

Relaxation and Fluctuations of Membrane Currents that Flow through Drug-Operated Channels

Proliferating cells, including cancer cells, require altered metabolism to efficiently incorporate nutrients such as glucose into biomass. The M2 isoform of pyruvate kinase (PKM2) promotes the metabolism of glucose by aerobic glycolysis and contributes to anabolic metabolism. Paradoxically, decreased pyruvate kinase enzyme activity accompanies the expression of PKM2 in rapidly dividing cancer cells and tissues. We demonstrate that phosphoenolpyruvate (PEP), the substrate for pyruvate kinase in cells, can act as a phosphate donor in mammalian cells because PEP participates in the phosphorylation of the glycolytic enzyme phosphoglycerate mutase (PGAM1) in PKM2-expressing cells. We used mass spectrometry to show that the phosphate from PEP is transferred to the catalytic histidine (His11) on human PGAM1. This reaction occurred at physiological concentrations of PEP and produced pyruvate in the absence of PKM2 activity. The presence of histidine-phosphorylated PGAM1 correlated with the expression of PKM2 in cancer cell lines and tumor tissues. Thus, decreased pyruvate kinase activity in PKM2-expressing cells allows PEP-dependent histidine phosphorylation of PGAM1 and may provide an alternate glycolytic pathway that decouples adenosine triphosphate production from PEP-mediated phosphotransfer, allowing for the high rate of glycolysis to support the anabolic metabolism observed in many proliferating cells.

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Evidence for an Alternative Glycolytic Pathway in Rapidly Proliferating Cells

The paper reports a study of the kinetics of the reaction between phosphoenolpyruvate, ADP and Mg(2+) catalysed by rabbit muscle pyruvate kinase. The experimental results indicate that the reaction mechanism is equilibrium random-order in type, that the substrates and products are phosphoenolpyruvate, ADP, Mg(2+), pyruvate and MgATP, and that dead-end complexes, between pyruvate, ADP and Mg(2+), form randomly and exist in equilibrium with themselves and other substrate complexes. Values were determined for the Michaelis, dissociation and inhibition constants of the reaction and are compared with values ascertained by previous workers.

/pdf/a-kinetic-study-of-rabbit-muscle-pyruvate-kinase-40fogyoqcw.pdf

A kinetic study of rabbit muscle pyruvate kinase.

The binding of aromatic sulphonic acids to bovine serum albumin

The effects that the environment exerts on the spectroscopic properties of certain dyes that are bound by bovine serum albumin.

Kinetic Studies on the Mechanism of the Malate Dehydrogenase Reaction

The paper reports a study of the kinetics of the reaction between phosphoenolpyruvate, ADP and Mg2+ catalysed by yeast pyruvate kinase when activated by fructose 1,6-diphosphate and K+. The experimental results indicate that the reaction mechanism is of the Ordered Tri Bi type with the substrates binding in the order phosphoenolpyruvate, ADP and Mg2+. Direct phosphoryl transfer takes place in the quaternary complex, with pyruvate released before MgATP. A dead-end enzyme–pyruvate complex is also indicated. Values have been determined for the Michaelis, dissociation and inhibition constants of the reaction. Several of the rate constants involved have also been evaluated.

/pdf/a-kinetic-study-of-baker-s-yeast-pyruvate-kinase-activated-4ugntzf2vl.pdf

Stanley Ainsworth

Papers

A kinetic study of rabbit muscle pyruvate kinase.

The binding of aromatic sulphonic acids to bovine serum albumin

The effects that the environment exerts on the spectroscopic properties of certain dyes that are bound by bovine serum albumin.

Kinetic Studies on the Mechanism of the Malate Dehydrogenase Reaction

A kinetic study of Baker's-yeast pyruvate kinase activated by fructose 1,6-diphosphate.