Arnold Boiteux

Allosteric properties of yeast pyruvate decarboxylase

Pyruvate decarboxylase (EC 4.1.1.1) of yeast was discovered by Neuberg and Karczag [l] and subsequently isolated and partially purified by several groups of investigators [2,3]. The authors reported, that the overall kinetics of the enzyme follow the Michaelis-Menten equation. However, as pointed out earlier [4] in studies on the control mechanism of glycolysis we found that the enzyme has allosteric properties. We here report the results of kinetic measurements carried out with cell-free extract and highly purified yeast enzyme.

Creative functions of instability and oscillations in metabolic systems.

Publisher Summary This chapter discusses the creative functions of instability and oscillations in metabolic systems. The aim of the biological regulatory mechanisms is to stabilize the important metabolic parameters in living organisms. Recognition of allosteric properties of regulatory enzymes has stimulated discussion of cell regulatory mechanisms in relation to cell homeostasis. The chapter focuses on the creative force of instability in regard to homeostasis and homogeneity as a new approach to the understanding of cellular regulation. Homeostasis in time and homogeneity in space constitute the classic frame work for a presentation of function and structure. Although exogenous fluctuations are filtered out by living systems, autonomous oscillations as well as dynamic in-homogeneity of matter could be observed in cells and multicellular bodies. It means that the organisms create their own timing and spacing, which might couple to external signals. The theoretical and experimental data reviewed allow concluding that the instability of metabolic systems may be important for the temporal and spatial organization of living matter.

SUBSTRATE CONTROL OF GLYCOLYTIC OSCILLATIONS

Publisher Summary Biochemical systems, like any other high order system, can rise to a specific level of activity and break into continuous oscillations if proper parametric conditions are maintained. Recent investigations in a number of laboratories revealed that the classical pathway of energy metabolism, namely glycolysis, offers an example of a biochemical network involving multiple feedback interactions and capable of continuous oscillatory performance. The relative completeness of the knowledge of the macroscopic features of the glycolytic enzymes, coenzymes, and intermediates allow a reasonable analysis and interpretation of the conditions and mechanisms yielding oscillations of glycolysis. This chapter discusses the relationship between the outer parameters of glycolysis, namely its substrates and the formal character of the glycolytic oscillations. It discusses the internal mechanism of the oscillation. An injection technique is used to allow a continuous and controlled input of glycolytic substrates and intermediates for the induction of reproducible steady states of the pathway.

Oscillations in glycolysis, cellular respiration and communication

In an attempt to understand oscillatory phenomena in chemical, subcellular and cellular systems, the mechanisms of oscillation as well as the general dynamics of glycolysis, cellular respiration and cyclic-AMP-controlled oscillation in the slime mould Dictyostelium discoideum have been studied. The enzymic sources of the oscillations have been identified with glycolysis as well as for the slime mould, and appropriate mathematical models have been developed. The primary reaction mechanism of mitochondrial oscillations is not yet known in detail. However, the studies show unequivocally that the conditions for the generation of oscillations can be summarized for all cases in four common statements:

CONTROL MECHANISM OF GLYCOLYTIC OSCILLATIONS

Publisher Summary Glycolytic self-excitation is produced by the allosteric oscillophor phosphofructokinase and propagated with a dependent phase shift by means of the coupling variable adenosine phosphates via the kinases, phosphoglycerate kinase and pyruvate kinase. The basis of a general mechanism of glycolytic oscillation is the establishment of a metabolite and energy balance of the pathway. Titration experiments with the oscillating system, as well as with isolated enzyme preparations, prove that the adenosine phosphate control variable is operating via the kinases of the glycolytic pathway. Glycolysis is controlled at any time by the adenosine phosphate variable. The variable is a function of the velocities of all adenosine triphosphate (ATP)-generating and ATP-splitting reactions and of the adenosine phosphate pool. Any experimental change of the ATP-splitting activities or adenosine phosphate pool influences the phase angle between the oscillophor and secondary oscillations of the other glycolytic intermediates. This has been tested under a great variety of conditions in the laboratory.

Circuit analysis of the oscillatory state in glycolysis

The oscillatory state of glycolysis in yeast extracts has been analysed by methods known from electronic circuit studies. The time course of the reactions are calculated by the method of least squares from experimentally determined sets of the concentrations of most of the metabolites. The dynamics of the glycolytic network of reactions can then be represented in terms of flow versus driving force (current versus voltage in the corresponding electronic circuit). The analysis of the dynamics leads to the conclusion that glycolysis is switched on and off in a pulsed manner during the oscillatory state. The resulting pulsed flow cannot only be measured with glycolytic end products, like carbon dioxide or ethanol, but can also readily be demonstrated by diagrams of reaction rates of single enzymic steps even in the initial stages of the glycolytic sequence. An analytic method widely applied to electronic circuits also proved to be useful in the study of the dynamics of a complex enzymic network.

Oscillatory phenomena in biological systems

Recently, under the auspices of the European Molecular Biology Organization (EMBO) and supported by the Max-Planck-Gesellschaft and the Deutsche Forschungsgemeinschaft, a workshop on oscillatory phenomena was held, 3-6 October 1976, covering the thermodynamic and kinetic requirements for the generation of periodic phenomena, mathematical methods as well as specific chemical, biochemical and biological systems.

Pattern Formation and Marangoni Convection during Oscillating Glycolysis

Abstract Spatial pattern formation during oscillating glycolysis in a thin layer of extract from yeast cells is investigated by two-dimensional spectrophotometry using video techniques and by dark-field detection of refractive index gradients. The time intervals of repetitively reappearing trans mission patterns coincide with the phases of maximum metabolic turnover during each glycolytic cycle. The spatial arrangement of the patterns is strongly correlated with a network of convection cells which permanently exists in the liquid layer. The patterns are generated by the coupling of reaction with Marangoni type convection due to gradients in surface tension which are mainly caused by evaporative cooling and influenced by chemical composition and reactivity.

Applications of Progress Curves in Enzyme Kinetics

Measuring progress curves and analyzing the data with elaborate numerical tools is a powerful technique in enzyme kinetics. The experience with this technique gained from the investigation of seven non-cooperative as well as cooperative enzymes is presented.