Phosphorylative allosteric

Figure 6.24 The function of the enzyme phosphofructokinase. (a) Phosphofructokinase is a key enzyme in the gycolytic pathway, the breakdown of glucose to pyruvate. One of the end products in this pathway, phosphoenolpyruvate, is an allosteric feedback inhibitor to this enzyme and ADP is an activator, (b) Phosphofructokinase catalyzes the phosphorylation by ATP of fructose-6-phosphate to give fructose-1,6-bisphosphate. (c) Phosphoglycolate, which has a structure similar to phosphoenolpyruvate, is also an inhibitor of the enzyme.

Muscle glycogen phosphorylase is a dimer of two identical subunits (842 residues, 97.44 kD). Each subunit contains a pyridoxal phosphate cofactor, covalently linked as a Schiff base to Lys °. Each subunit contains an active site (at the center of the subunit) and an allosteric effector site near the subunit interface (Eigure 15.15). In addition, a regulatory phosphorylation site is located at Ser on each subunit. A glycogen-binding site on each subunit facilitates prior association of glycogen phosphorylase with its substrate and also exerts regulatory control on the enzymatic reaction. 

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. 

Pyruvate kinase possesses allosteric sites for numerous effectors. It is activated by AMP and fructose-1,6-bisphosphate and inhibited by ATP, acetyl-CoA, and alanine. (Note that alanine is the a-amino acid counterpart of the a-keto acid, pyruvate.) Furthermore, liver pyruvate kinase is regulated by covalent modification. Flormones such as glucagon activate a cAMP-dependent protein kinase, which transfers a phosphoryl group from ATP to the enzyme. The phos-phorylated form of pyruvate kinase is more strongly inhibited by ATP and alanine and has a higher for PEP, so that, in the presence of physiological levels of PEP, the enzyme is inactive. Then PEP is used as a substrate for glucose synthesis in the pathway (to be described in Chapter 23), instead... 

Johnson, L. N., 1992. Glycogen phosphorylase Control by phosphorylation and allosteric effectors. FASEB Journal 6 2274-2282. 

Regulatory Control of Fatty Acid Metabolism—An Interplay of Allosteric Modifiers and Phosphorylation-Dephosphorylation Cycles... 

The first pharmacological agent shown to activate AMPK was 5-aminoimidazole-4-carboxamide (AICA) riboside, also known as acadesine. This adenosine analogue is taken up into cells by adenosine transporters and phosphoiylated by adenosine kinase to the mono-phosphorylated form, AICA ribotide or ZMP. ZMP accumulates inside cells to higher concentrations than the concentration of AICA riboside present in the medium, and it mimics both effects of AMP on AMPK system (allosteric activation and inhibition of... 

Protein phosphorylation-dephosphorylation is a highly versatile and selective process. Not all proteins are subject to phosphorylation, and of the many hydroxyl groups on a protein s surface, only one or a small subset are targeted. While the most common enzyme function affected is the protein s catalytic efficiency, phosphorylation can also alter the affinity for substrates, location within the cell, or responsiveness to regulation by allosteric ligands. Phosphorylation can increase an enzyme s catalytic efficiency, converting it to its active form in one protein, while phosphorylation of another converts it into an intrinsically inefficient, or inactive, form (Table 9—1). 

Many proteins can be phosphorylated at multiple sites or are subject to regulation both by phosphorylation-dephosphorylation and by the binding of allosteric ligands. Phosphorylation-dephosphorylation at any one site can be catalyzed by multiple protein kinases or protein phosphatases. Many protein kinases and most protein phosphatases act on more than one protein and are themselves interconverted between active and inactive forms by the binding of second messengers or by covalent modification by phosphorylation-dephosphorylation. 

This reaction is followed by another phosphorylation with ATP catalyzed by the enzyme phosphofructoki-nase (phosphofructokinase-1), forming fructose 1,6-bisphosphate. The phosphofructokinase reaction may be considered to be functionally irreversible under physiologic conditions it is both inducible and subject to allosteric regulation and has a major role in regulating the rate of glycolysis. Fructose 1,6-bisphosphate is cleaved by aldolase (fructose 1,6-bisphosphate aldolase) into two triose phosphates, glyceraldehyde 3-phosphate and dihydroxyacetone phosphate. Glyceraldehyde 3-phosphate and dihydroxyacetone phosphate are inter-converted by the enzyme phosphotriose isomerase. 

The principal enzymes controlling glycogen metabolism—glycogen phosphorylase and glycogen synthase— are regulated by allosteric mechanisms and covalent modifications due to reversible phosphorylation and... 

Muscle phosphorylase is distinct from that of Hver. It is a dimer, each monomer containing 1 mol of pyridoxal phosphate (vitamin Bg). It is present in two forms phos-phoiylase a, which is phosphorylated and active in either the presence or absence of 5 -AMP (its allosteric modifier) and phosphorylase h, which is dephosphorylated and active only in the presence of 5 -AMP. This occurs during exercise when the level of 5 -AMP rises, providing, by this mechanism, fuel for the muscle. Phosphorylase a is the normal physiologically active form of the enzyme. 

Ghanges in the availability of substrates are responsible for most changes in metabolism either directly or indirectly acting via changes in hormone secretion. Three mechanisms are responsible for regulating the activity of enzymes in carbohydrate metabolism (1) changes in the rate of enzyme synthesis, (2) covalent modification by reversible phosphorylation, and (3) allosteric effects. 

Fructose 2,6-bisphosphate is formed by phosphorylation of fructose 6-phosphate by phosphofructoki-nase-2. The same enzyme protein is also responsible for its breakdown, since it has fructose-2,6-hisphos-phatase activity. This hifrmctional enzyme is under the allosteric control of fructose 6-phosphate, which stimulates the kinase and inhibits the phosphatase. Hence, when glucose is abundant, the concentration of fructose 2,6-bisphosphate increases, stimulating glycolysis by activating phosphofructokinase-1 and inhibiting... 

Acetyl-CoA carboxylase is an allosteric enzyme and is activated by citrate, which increases in concentration in the well-fed state and is an indicator of a plentiful supply of acetyl-CoA. Citrate converts the enzyme from an inactive dimer to an active polymeric form, having a molecular mass of several milhon. Inactivation is promoted by phosphorylation of the enzyme and by long-chain acyl-CoA molecules, an example of negative feedback inhibition by a product of a reaction. Thus, if acyl-CoA accumulates because it is not esterified quickly enough or because of increased lipolysis or an influx of free fatty acids into the tissue, it will automatically reduce the synthesis of new fatty acid. Acyl-CoA may also inhibit the mitochondrial tricarboxylate transporter, thus preventing activation of the enzyme by egress of citrate from the mitochondria into the cytosol. 

Lipogenesis is regulated at the acetyl-CoA carboxylase step by allosteric modifiers, phosphorylation/de-phosphorylation, and induction and repression of enzyme synthesis. Citrate activates the enzyme, and long-chain acyl-CoA inhibits its activity. Insulin activates acetyl-CoA carboxylase whereas glucagon and epinephrine have opposite actions. 

Synthetic nonhydrolyzable analogs of nucleoside triphosphates (Figure 33-13) allow investigators to distinguish the effects of nucleotides due to phosphoryl transfer from effects mediated by occupancy of allosteric nucleotide-binding sites on regulated enzymes. 

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