Citrate phosphofructokinase

Glycolysis and the citric acid cycle (to be discussed in Chapter 20) are coupled via phosphofructokinase, because citrate, an intermediate in the citric acid cycle, is an allosteric inhibitor of phosphofructokinase. When the citric acid cycle reaches saturation, glycolysis (which feeds the citric acid cycle under aerobic conditions) slows down. The citric acid cycle directs electrons into the electron transport chain (for the purpose of ATP synthesis in oxidative phosphorylation) and also provides precursor molecules for biosynthetic pathways. Inhibition of glycolysis by citrate ensures that glucose will not be committed to these activities if the citric acid cycle is already saturated. 

Figure 5.3 Major control points of glycolysis and the TCA cycle. Enzymes I, hexokinase II, phosphofructokinase III, pyruvate kinase IV, pyruvate dehydrogenase V, citrate synthase VI, aconitase VII, isocitrate dehydrogenase VIII, a-oxoglutarate dehydrogenase.

Phosphofructokinase-1 T T Insulin Glucagon (cAMP) AMP, fructose 6-phosphate, P, fructose 2,6-bisphos-phate Citrate (fatty acids, ketone bodies), ATP, glucagon (cAMP)... 

Phosphofructokinase (PFK) is a key regulatory enzyme of glycolysis that catalyzes the conversion of fructose-6-phosphate to fructose-1,6-diphosphate. The active PFK enzyme is a homo- or heterotetrameric enzyme with a molecular weight of 340,000. Three types of subunits, muscle type (M), liver type (L), and fibroblast (F) or platelet (P) type, exist in human tissues. Human muscle and liver PFKs consist of homotetramers (M4 and L4), whereas red blood cell PFK consists of five tetramers (M4, M3L, M2L2, ML3, and L4). Each isoform is unique with respect to affinity for the substrate fructose-6-phosphate and ATP and modulation by effectors such as citrate, ATP, cAMP, and fructose-2,6-diphosphate. M-type PFK has greater affinity for fructose-6-phosphate than the other isozymes. AMP and fructose-2,6-diphosphate facilitate fructose-6-phosphate binding mainly of L-type PFK, whereas P-type PFK has intermediate properties. 

Substrates can affect the conformation of the other active sites. So can other molecules. Effector molecules other than the substrate can bind to specific effector sites (different from the substrate-binding site) and shift the original T-R equilibrium (see Fig. 8-9). An effector that binds preferentially to the T state decreases the already low concentration of the R state and makes it even more difficult for the substrate to bind. These effectors decrease the velocity of the overall reaction and are referred to as allosteric inhibitors. An example is the effect of ATP or citrate on the activity of phosphofructokinase. Effectors that bind specif-... 

Phosphofructokinase-1 Fructose 6-phosphate AMP Fructose 2,6-bisphosphate Phosphoenolpyruvate ATP Citrate ... 

Phosphofructokinase-1 is also inhibited by citrate, the first intermediate of the citric acid cycle. When the cycle is idling, citrate accumulates within mitochondria, then spills into the cytosol. When the concentrations of both ATP and citrate rise, they produce a concerted allosteric inhibition of phosphofructokinase-1 that is greater than the sum of their individual effects, slowing glycolysis. 

Heterotropic effectors The effector may be different from the substrate, in which case the effect is said to be heterotropic. For example, consider the feedback inhibition shown in Figure 5.17. The enzyme that converts A to B has an allosteric site that binds the end-product, E. If the concentration of E increases (for example, because it is not used as rapidly as it is synthesized), the initial enzyme in the pathway is inhibited. Feedback inhibition provides the cell with a product it needs by regulating the flow of substrate molecules through the pathway that synthesizes that product. [Note Heterotropic effectors are commonly encountered, for example, the glycolytic enzyme phosphofructokinases allosterically inhibited by citrate, which is not a substrate for the enzyme (see p. 97).]... 

The reaction catalyzed by phosphofructokinase A. is activated by high concentrations of ATP and citrate. B. uses fructose 1-phosphate as substrate. C. is the regulated reaction of the glycolytic pathway. D. is near equilibrium in most tissues. E. is inhibited by fructose 2,6-bisphosphate. Correct answer = C. Phosphofructokinase is the pace-setting enzyme of glycolysis. It is inhibited by ATP and citrate, uses fructose 6-phosphate as substrate, and catalyzes a reaction that is far from equilibrium. The reaction is activated by fructose 2,6-bisphosphate. 

It is now generally recognized that an important site of regulation of both glycolysis and gluconeogenesis is at the level of fructose diphosphate formation and hydrolysis (10). In the direction of glycolysis, the activity of phosphofructokinase is inhibited by ATP and citrate, and this inhibition is reversed by AMP (11). The discovery that FDPase... 

In addition to its importance in providing cytosolic acetyl-CoA and NADPH, citrate also serves as a major regulator of the rate of fatty acid synthesis. As we shall see (chapter 18) citrate is a strong positive modifier of the first reaction in fatty acid synthesis. It should be remembered (see chapter 12) that citrate also is a negative modifier of phosphofructokinase and thereby exerts a negative effect on glycolysis, which also occurs in the cytosol. 

Newsholme E. A., Sugden P. H. and Williams T. (1977) Effect of citrate on the activities of 6-phosphofructokinase from nervous and muscle tissues from different animals and its relationships to the regulation of glycolysis. Biochem. J. 166, 123-129. 

In liver phosphofructokinase, ATP, ADP, and citrate are effectors of the reaction rate (see Fig. 9-18). Define what types of effectors they are. 

Question Citrate, NADH, and long-chain fatty acids inhibit the activity of phosphofructokinase. From where do these effectors arise, and what is the significance of their action ... 

Fructose 1,6-bisphosphatase is inhibited by AMP but activated by citrate and 3-phosphoglycerate. Thus, in a high-bond-energy state, an increase of citrate and a decrease of AMP combine to activate fructose-1,6-bisphosphatase and to inhibit phosphofructokinase (Fig. 11-10). This promotes the hydrolysis of fructose 1,6-bisphosphate to fructose 6-phosphate. 

Figure 16.17. Allosteric Regulation of Phosphofructokinase. A high level of ATP inhibits the enzyme by decreasing its affinity for fructose 6-phosphate. AMP diminishes and citrate enhances the inhibitory effect of ATP. 

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