Long term oscillation in glycolysis

To increase the period of glycolytic oscillations in yeast extracts (Saccharomyces uvarum), the dependence of the period on pH, on concentrations of phosphate and enzymes, and on temperature has been studied. Stable oscillatory trans were obtained at a pH value of about 6.5. Increasing the phosphate and decreasing the enzyme concentrations as well as decreasing temperature lengthened the period. By dilution of the extract with buffer while maintaining the metabolite concentrations at their initial level the period could be successively prolonged from 20 min to about 6 h.     

Oscillations and efficiency in glycolysis

We suggest that temporal oscillations of concentrations of intermediates in biochemical reaction systems may enhance the efficiency of free energy conversion (reduce dissipation) in those reactions. Experiments on glycolysis are used to estimate the Gibbs free energy changes along the glycolysis mechanism, and to postulate a construct for the glycolysis "machine" which involves: the PFK reaction as the primary oscillophor; the GAPDH reaction as a phase-shifting device; and the PK reaction with the property of intrinsic oscillatory response at resonance with the driving frequency. Analysis of a simple reaction mechanism with these postulated properties shows that the conversion of free energy from reactants to products is more efficient in an oscillatory than a steady state operation. The efficiency of free energy conversion in glycolysis from glucose + ADP to products + ATP is estimated to be increased by 5--10% due to oscillations. This may have been relevant for the evolutionary development of oscillations such as in glycolysis, especially in anaerobic cells.     

Design of glycolysis

The design of the glycolytic pathway resulting from the continuous refinement of evolution is discussed with regard to three aspects. 1. Functional and structural properties of individual enzymes. The catalytic constants of the glycolytic enzymes are remarkably optimized; the turnover numbers are within one order of magnitude. The same is true for the molarities of catalytic centres in the cytosol, as is noted for yeast. Functional properties of the enzymes are reflected in their tertiary and quaternary structures. 2. Regulatory mechanisms of single enzymes. A classification of the various types of enzymic control mechanisms operating in the glycolytic pathway is given. In addition to the usual Michaelis-Menten saturation kinetics and the various types of inhibition there is control by positive and negative effectors based on oligomeric structures (fast acting, fine control) as well as regulation by chemical interconversion structures (fast acting, fine control) as well as regulation by chemical based on enzymes cascades (slow acting, very effective). 3. Functional and regulatory mechanisms of the whole glycolytic reaction pathway. A prominent feature is the high enzyme:substrate ratio, which guarantees fast response times. However, a quantitative treatment of the overall kinetics is limited by an incomplete knowledge of the enzymes' dynamic and chemical compartmentation as well as some of their control properties. From an analysis of the oscillatory state, certain control points in the glycolytic chain can be located that coincide with major branching points to other metabolic pathways. These points are controlled by fast-acting cooperative enzymes that operate in a flip-flop mechanism together with the respective antagonistic enzymes, preventing futile cycles. The gating enzymes leading to the glycogen store and the citric acid cycle are of the slow-acting but very effective interconvertible type. The combination of all the complex and intricate features of design yields a glycolytic network that enables the cell to respond to its various metabolic needs quickly, effectively and economically.     

Modeling cancer glycolysis

Most cancer cells exhibit an accelerated glycolysis rate compared to normal cells. This metabolic change is associated with the over-expression of all the pathway enzymes and transporters (as induced by HIF-1α and other oncogenes), and with the expression of hexokinase (HK) and phosphofructokinase type 1 (PFK-1) isoenzymes with different regulatory properties. Hence, a control distribution of tumor glycolysis, modified from that observed in normal cells, can be expected. To define the control distribution and to understand the underlying control mechanisms, kinetic models of glycolysis of rodent AS-30D hepatoma and human cervix HeLa cells were constructed with experimental data obtained here for each pathway step (enzyme kinetics; steady-state pathway metabolite concentrations and fluxes). The models predicted with high accuracy the fluxes and metabolite concentrations found in living cancer cells under physiological O(2) and glucose concentrations as well as under hypoxic and hypoglycemic conditions prevailing during tumor progression. The results indicated that HK≥HPI>GLUT in AS-30D whereas glycogen degradation≥GLUT>HK in HeLa were the main flux- and ATP concentration-control steps. Modeling also revealed that, in order to diminish the glycolytic flux or the ATP concentration by 50%, it was required to decrease GLUT or HK or HPI by 76% (AS-30D), and GLUT or glycogen degradation by 87-99% (HeLa), or decreasing simultaneously the mentioned steps by 47%. Thus, these proteins are proposed to be the foremost therapeutic targets because their simultaneous inhibition will have greater antagonistic effects on tumor energy metabolism than inhibition of all other glycolytic, non-controlling, enzymes.     

Reversal of glycolysis in yeast.

When a buffered, aerobic suspension of ethanol-grown cells of Saccharomyces cerevisiae is treated with ethanol, a rapid flux of metabolism is observed from endogenous phosphoenolpyruvate to hexose monophosphates. Intracellular concentrations of phosphoenolpyruvate, 2-phosphoglycerate, and 3-phosphoglycerate record a monotonic drop, while those of triose phosphates and fructose 1,6-diphosphate fall after an early rise; fructose 6-phosphate, mannose 6-phosphate, and glucose 6-phosphate levels rise to a plateau. Prior growth on glucose extinguishes fructose 1,6-diphosphatase activity and completely arrests the rise of the hexose monophosphates. By using mutants blocked at a number of glycolytic steps it has been concluded that the metabolic flow takes place along the Embden-Meyerhof pathway in the reverse direction bypassing pyruvate kinase and fructose 6-phosphate kinase. Ethanol acts as a trigger by supplying NADH at the glyceraldehyde 3-phosphate dehydrogenase step. The rate of the reversal in the span phosphoenolpyruvate to fructose 1,6-diphosphate approaches 40 μ mol of 3-carbon units per minute per gram of wet cells. The in vivo activity of fructose 1,6-diphosphatase is nearly a quarter of this rate.     

A futile cycle in erythrocyte glycolysis

The storage lesion which limits the shelf life of human blood in blood banking is associated with a metabolic loss of 2,3-diphosphoglycerate and ATP. This metabolic loss is driven by intracellular ATPases which are usually considered to include the ion pumps and the reactions which maintain the discoid shape of the human erythrocyte. Under the acidic conditions of blood storage, the energy-yielding reactions of the glycolytic pathway are restricted at the hexokinase and phosphofructokinase steps. We show here that under such circumstances the enzyme of the diphosphoglycerate shunt, diphosphoglycerate mutase/phosphatase and the glycolytic enzyme phosphoglycerate kinase can form a futile cycle with ATPase activity. This ATPase activity responds to 2-phosphoglycolate which is known to activate both diphosphoglycerate mutase and diphosphoglycerate phosphatase reactions. When the enzymes of the futile cycle are combined with the enzymes of the lower glycolytic pathway in a reconstitution experiment designed to represent conditions within the stored erythrocyte, the futile cycle does provide an ATPase activity which results in the metabolic loss of 2,3-diphosphoglycerate. An isotope incorporation experiment demonstrates that the futile cycle is active in glucose-depleted erythrocytes.