Pyruvate kinase, enzymic activity

Pyruvate kinase the last enzyme in aerobic glycolysis, it catalyzes a substrate-level phosphorylation of ADP using the high-energy substrate phosphoenolpyruvate (PEP). Pyruvate kinase is activated by fructose 1,6-bisphosphate from the PFK-1 reaction (feedforward activation). 

The key regulatory enzymes in glycolysis are phosphory-lase, hexokinase, phosphofructokinase and pyruvate kinase, the activities of which are stimulated by the increase in the concentrations of AMP and phosphate and the decrease in that of phosphocreatine. These mechanisms are discussed in Chapters 6 and 9 Figures 6.16 and 9.27. 

J2. Jemelin, M., Fornerod, M., Frei, J., and Prod hom, L. S., Impaired phagocytosis in leukocytes from newborn infants. A study of glycolysis and activities of phosphoglycerate kinase and pyruvate kinase. Enzyme 12, 642-646 (1971). 

Pyruvate kinase (PK) activity in hemolyzed erythrocytes has been determined by using an LDH-lactate monooxygenase sequence electrode (Weigelt et al., 1988). The enzymes were immobilized in gelatin and attached to an oxygen probe. Since the sample material contains only... 

Another activator of pyruvate kinase enzyme is 6-phosphogluconate, which is formed in the pentosphosphate pathway. The pentosphosphate pathway is particularly active in the liver during conditions of fatty-acid synthesis. The 6-phosphogluconate may well be an intervening signal between the pentosphosphate pathway and glycolysis because an increased activity of pyruvate... 

The PCK of A. succinidproducens requires the nucleoside triphosphate ADP, and a pyruvate kinase-like activity of this enzyme was identified. In gluconeogenesis, PCK catalyzes the irreversible decarboxylation of oxalacetate, and in A. succinici-producens, AMP was shown to activate this oxalacetate decarboxylase reaction (Jabalquinto et al. 1999). 

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... 

Pyruvate kinase (PK) is one of the three postulated rate-controlling enzymes of glycolysis. The high-energy phosphate of phosphoenolpyruvate is transferred to ADP by this enzyme, which requires for its activity both monovalent and divalent cations. Enolpyruvate formed in this reaction is converted spontaneously to the keto form of pyruvate with the synthesis of one ATP molecule. PK has four isozymes in mammals M, M2, L, and R. The M2 type, which is considered to be the prototype, is the only form detected in early fetal tissues and is expressed in many adult tissues. This form is progressively replaced by the M( type in the skeletal muscle, heart, and brain by the L type in the liver and by the R type in red blood cells during development or differentiation (M26). The M, and M2 isozymes display Michaelis-Menten kinetics with respect to phosphoenolpyruvate. The Mj isozyme is not affected by fructose-1,6-diphosphate (F-1,6-DP) and the M2 is al-losterically activated by this compound. Type L and R exhibit cooperatively in... 

In biological systems, therefore, the behavior of Li+ is predicted to be similar to that of Na+ and K+ in some cases, and to that of Mg2+ and Ca2+ in others [12]. Indeed, research has demonstrated numerous systems in which one or more of these cations is normally intrinsically involved, including ion transport pathways and enzyme activities, in which Li+ has mimicked the actions of these cations, sometimes producing inhibitory or stimulatory effects. For example, Li+ can replace Na+ in the ATP-dependent system which controls the transport of Na+ through the endoplasmic reticulum Li+ inhibits the activity of some Mg2+-dependent enzymes in vitro, such as pyruvate kinase and inositol monophosphate phosphatase Li+ affects the activity of some Ca2+-dependent enzymes— it increases the levels of activated Ca2+-ATPase in human erythrocyte membranes ex vivo and inhibits tryptophan hydroxylase. 

In addition to the aforementioned allenic steroids, prostaglandins, amino acids and nucleoside analogs, a number of other functionalized allenes have been employed (albeit with limited success) in enzyme inhibition (Scheme 18.56) [154-159]. Thus, the 7-vinylidenecephalosporin 164 and related allenes did not show the expected activity as inhibitors of human leukocyte elastase, but a weak inhibition of porcine pancreas elastase [156], Similarly disappointing were the immunosuppressive activity of the allenic mycophenolic acid derivative 165 [157] and the inhibition of 12-lipoxygenase by the carboxylic acid 166 [158]. In contrast, the carboxyallenyl phosphate 167 turned out to be a potent inhibitor of phosphoenolpyruvate carboxylase and pyruvate kinase [159]. Hydrolysis of this allenic phosphate probably leads to 2-oxobut-3-enoate, which then undergoes an irreversible Michael addition with suitable nucleophilic side chains of the enzyme. 

Feed-forward control is more likely to be focused on a reaction occurring at or near the end of a pathway. Compounds produced early in the pathway act to enhance the activity of the control enzyme and so prevent a back log of accumulated intermediates just before the control point. An example of feed-forward control is the action of glucose-6-phosphate, fructose-1,6-bisphosphate (F-l,6bisP) and phosphoenol pyruvate (PEP), all of which activate the enzyme pyruvate kinase in glycolysis in the liver. 

Control of pymvate dehydrogenase activity is via covalent modification a specific kinase causes inactivation of the PDH by phosphorylation of three serine residues located in the pyruvate decarboxylase/dehydrogenase component whilst a phosphatase activates PDH by removing the phosphates. The kinase and phosphatase enzymes are non-covalently associated with the transacetylase unit of the complex. Here again we have an example of simultaneous but opposite control of enzyme activity, that is, reciprocal regulation. 

Two key enzymes in the pathway are regnlated by interconversion cycles they are the regnlatory enzyme PFK-2, and the glycolytic enzyme pyruvate kinase. There are two separate protein kinases that phosphorylate these enzymes and they both resnlt in activation of these enzymes. Dephosphorylation inactivates them. 

Adrenaline increases the rate of gluconeogenesis it binds to the a-receptor on the surface of the liver cell, which results in an increase in cytosolic concentration of Ca " ions (Chapter 12). This increases the activity of the Ca " -catmodulin-dependent protein kinase which phosphory-lates and causes similar changes in the activities of the enzymes PFK-2 and pyruvate kinase to those resulting from activation of cyclic-AMP-dependent protein kinase. Hence Ca " ions increase the rate of gluconeogenesis. 

The hormones glucagon, cortisol and insulin regulate the concentrations of some enzymes and hence their activities. These include glucokinase, pyruvate kinase and phospho-enolpyruvate carboxykinase. Most work has been carried out on the carboxykinase enzyme, for which it is known that glucagon and cortisol increase the concentration whereas insulin decreases it. These changes are brought about at the transcriptional level by changing the activity of transcription factors (Chapter 20). Since the hormones... 

A regulatory effect observed in many biochemical pathways an enzyme, which catalyzes a step (typically a late step) in a pathway, is activated by elevated levels of a precursor of a substrate for that enzyme. A possible example of feed-forward activation may be the action of elevated levels of fructose 1,6-bisphosphate on pyruvate kinase. 

Nucleotide Base Conformation. Using NMR data, a relationship between the degree of specificity and the conformation of bound ATP at the active site has been shown for a number of ATP utilizing enzymes. Two examples of these are cAMP-dependent protein kinase and pyruvate kinase (18,19) It appears that enzymes that exhibit higher nucleotide triphosphate specificity bind ATP so... 

Affinity Labeling of Catalytic ATP Sites. Residues involved in ATP binding are potentially revealed by the use of affinity labels that are based on ATP s structure. Perhaps the most systematically studied of these compounds is 5 -fluorosulfonylbenzoyladenosine (5 -FSBA) (Figure 3a), which has been reported to label at least six kinases (32-A1). In the case of rabbit muscle pyruvate kinase such work has Indicated the presence of a tyrosine residue within the metal nucleotide binding site and an essential cysteine residue located at or near the free metal binding site (32). A similar reagent, 5 -FSBGuanosine, revealed the presence of two cysteine residues at the catalytic site of this same enzyme, both distinct residues from those modified by 5 -FSBA (33,34). With yeast pyruvate kinase both tyrosine and cysteine residues were modified by 5 -FSBA at the catalytic site ( ), and with porcine cAMP-dependent protein kinase a lysine residue was labeled at the active site (36). 

---