Effect of macromolecular crowding on the kinetics of glycolytic enzymes and the behaviour of glycolysis in yeast

Abstract

Water is involved in all aspects of biological activity, both as a solvent and as a reactant. It is hypothesized that intracellular water is in a highly structured state due to the high concentrations of macromolecules in the cell and that this may change the activity of intracellular enzymes due to altered binding affinities and allosteric regulations. Here we first investigate the kinetics of two glycolytic enzymes in artificially crowded aqueous solutions and show that crowding does indeed change their kinetics. Based on our kinetic measurements we propose a new model of oscillating glycolysis that instead of Michaelis-Menten or Monod-Wyman-Changeux kinetics uses the Yang-Ling adsorption isotherm introduced by G. Ling in the frame of the Association-Induction (AI) hypothesis. Using this model, we can reproduce previous experimental observations of the coupling of glycolytic oscillations and intracellular water dynamics, e.g., (i) during the metabolic oscillations, the latter variable oscillates in phase with ATP activity, and (ii) the emergence of glycolytic oscillations largely depends on the extent of intracellular water dipolar relaxation in cells in the resting state. Our results support the view that the extent of intracellular water dipolar relaxation is regulated by the ability of cytoplasmic proteins to polarize intracellular water with the assistance of ATP, as suggested in the AI hypothesis. This hypothesis may be relevant to the interpretation of many other biological oscillators, including cell signalling processes.

Coupled Response of Membrane Hydration with Oscillating Metabolism in Live Cells: An Alternative Way to Modulate Structural Aspects of Biological Membranes?

“…This has been substantiated by a large amount of experimental work performed in both in vitro and in vivo systems, suggesting that most of the intracellular water exists in a polarized state when the cell is in a resting state [18,19]. This view offers a reasonable explanation of our results on the oscillatory coupling between intracellular water relaxation (measured by the fluorescence response of a series of 6-acyl-2-(dimethylamino)naphtalene probes) and central metabolites (such as ATP) during glycolytic oscillations including its dependence on the integrity of the actin cytoskeleton [12,13,15]. For instance, using a mathematical model based on the Yang-Ling isotherm [22,23], which is grounded on the principles of the AIH, we were able to adequately reproduce the coupling between the oscillations of ATP and the intracellular water dynamics observed in our experiments [13,15].…”

“…This view offers a reasonable explanation of our results on the oscillatory coupling between intracellular water relaxation (measured by the fluorescence response of a series of 6-acyl-2-(dimethylamino)naphtalene probes) and central metabolites (such as ATP) during glycolytic oscillations including its dependence on the integrity of the actin cytoskeleton [12,13,15]. For instance, using a mathematical model based on the Yang-Ling isotherm [22,23], which is grounded on the principles of the AIH, we were able to adequately reproduce the coupling between the oscillations of ATP and the intracellular water dynamics observed in our experiments [13,15]. We also found that the Yang-Ling isotherm was more reliable in describing the kinetics of glycolytic enzymes in crowded environments than classical models based on mass action kinetics such as Michaelis-Menten (MM) and Monod-Changeux-Wyman (MCW) [15].…”

“…They also support the view of the cellular interior as a highly structured and near equilibrium system where energy inputs can be low and sustain regular oscillatory regimes. We argue that all of these experimental results [12,13,14,15], which are difficult to rationalize under the dominant view of the cell, are in line with the association-induction hypothesis (AIH) [17,18,19]. This theory, which still remains unrefuted, offers an alternative conceptual approach based on well-established principles of colloidal physical chemistry to explain how emergent features of the intracellular environment may give rise to cellular function.…”

“…For instance, using a mathematical model based on the Yang-Ling isotherm [22,23], which is grounded on the principles of the AIH, we were able to adequately reproduce the coupling between the oscillations of ATP and the intracellular water dynamics observed in our experiments [13,15]. We also found that the Yang-Ling isotherm was more reliable in describing the kinetics of glycolytic enzymes in crowded environments than classical models based on mass action kinetics such as Michaelis-Menten (MM) and Monod-Changeux-Wyman (MCW) [15]. The Yang-Ling isotherm is a general framework for models in the sense that it can generate both hyperbolic (MM) and sigmoidal (MCW) kinetics for single or multiple subunit enzymes [15].…”

“…In a series of papers [12,13,14,15], we reported a number of key observations that challenge some basic tenets of the established view of cellular systems. Particularly, we observed that during glycolytic oscillations, the extent of dipolar relaxation in the cellular interior, which is governed mainly by the rotational dynamics of intracellular water, is coupled to the behavior of the pathway (intervening metabolites such as ATP and NADH) in that it oscillates synchronously [12,13].…”