Biochemistry - Chemical Reactions in the Living Cell, Volume 2 - D. Metzler 1980

Biosynthesis: How New Molecules Are Formed
Regulation of Biosynthetic Processes
Phosphofructokinase: A Key Regulatory Enzyme

In most Cells, the metabolic interconversions of glucose-1-phosphate, glucose-6-phosphate, and fructose-6-phosphate either reach an equilibrium state or come very close to it. As for the phosphorylation of fructose-6-phosphate to fructose-1,6-diphosphate coupled with ATP (Fig. 11-11, top center), it typically falls far short of equilibrium. This fact was established by comparing the mass action ratio

Class="center">[Fructose diphosphate] [ADP]/[Fructose-6-phosphate] [ATP],

measured in Tissues with the known Equilibrium Constant for this reaction. At equilibrium, these two values should be equal (Section B,3). The experimental Methods for determining the four metabolic concentrations in tissues (which are necessary for estimating the mass action ratio) are of considerable interest, and we shall briefly review them. Tissues must be frozen extremely rapidly. Most commonly, this is achieved by pressing them between two massive aluminum plates cooled with liquid nitrogen¹). Using this method, tissues can be cooled down to —80 °C in less than 0.1 seconds. The frozen tissues are then pulverized, treated with a chilled protein-denaturing agent such as perchloric acid, and analyzed.

For the reaction catalyzed by phosphofructokinase, the mass action ratio in cardiac Muscle was found to be 0.03 [45], which is significantly lower than the equilibrium constant of 3000 (calculated from ∆G(pH7) = —20.1 kJ∙mol^-1). Since this practically irreversible reaction is far from equilibrium in tissues, phosphofructokinase can be considered the rate-limiting enzyme for overall Glycolysis (Chapter 6, Section E,1). Consistent with this role, phosphofructokinase is known to be sensitive to various allosteric inhibitors and activators (Fig. 11-11)²⁾. Phosphofructokinase is inhibited by high concentrations of ATP, whereas AMP exerts an activating effect.

Thus, a critical factor in The regulation of this enzyme, as well as many Other Enzymes involved in glycolysis and Gluconeogenesis, is the phosphorylation state of the adenylyl system. There is good reason to believe that AMP "switches on" this initial and most crucial step of glycolysis. The state of the adenylyl system also influences subsequent stages of glycolysis and The Tricarboxylic Acid Cycle. Consequently, a decrease in ATP concentration leads to the inhibition of Pyruvate and isocitrate oxidation. Furthermore, inorganic phosphate is required during the initial stage of Glycogen phosphorolysis and The oxidation of triose phosphates. Therefore, rapid cellular consumption of ATP (such as During Muscle contraction) results in a decreased ATP concentration and increased concentrations of AMP and P_i. All of these changes stimulate glycolysis. Conversely, when muscle activity ceases and ATP levels rise, multiple stages of glycolysis are simultaneously inhibited (Fig. 11-11).

¹⁾ For details, see Newsholme and Start [45].

²⁾ Among the allosteric inhibitors affecting this enzyme, zinc ions may be noted, with a binding constant of approximately 0.3 µM [46a].

FIG. 11-11. Interconnected pathways of glycolysis, gluconeogenesis, Fatty acid oxidation, and Biosynthesis, indicating various regulatory mechanisms: (→) Glycolytic and oxidative reactions proceeding via the tricarboxylic acid cycle. Solid bold arrows indicate the carbon pathway from glycogen (top right) to CO₂. (→) Biosynthetic pathways. Dashed bold arrows denote the gluconeogenic pathway from pyruvate via oxaloacetate and malate.