Pyruvate Phosphoenolpyruvate kinase

Termonia and Ross (1981-1,2) developed a reaction scheme coupling phosphofructo-kinase and pyruvate kinase reactions by referring to experimental observations of known activations and inhibitions of enzymes by metabolites. By numerical analysis of the rate equations they confirmed the oscillations in the concentrations of fructose-6-phosphate, pyruvate, phosphoenolpyruvate, fructose 1,6-biphosphate, and ADP. 

FIGURE 3.13 Phosphoenolpyruvate (PEP) is produced by the euolase reaction (hi glycolysis see Chapter 19) and hi turn drives the phosphorylation of ADP to form ATP in the pyruvate kinase reaction. 

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... 

The Jirst indirect route in glucose synthesis involves the formation of phosphoenolpyruvate from pyruvate without the intervention of pyruvate kinase. This route is catalyzed by two enzymes. At first, pyruvate is converted into oxaloacetate. This reaction occurs in the mitochondria as the pyruvate molecules enter them, and is catalyzed by pyruvate carboxylase according to the scheme... 

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. 

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 pyrnvate/phosphoenolpyrnvate cycle, which involves the enzymes pyrnvate kinase, pyruvate carboxylase and phosphoenolpyruvate carboxykinase. 

Hydroxycyclopropanecarboxylic acid phosphate HCP 34 is an analogue of phosphoenolpyruvate (PEP) 35 which is metabolized by various enzymes. HCP 34 is a potent competitive inhibitor of enzymes utilizing PEP 35, such as PEP carboxylase, enolase, pyruvate kinase, and probably other enzymes. It is a substantially better inhibitor than phospholactate 36 or phosphoglycolate 37, presumably because of the similarity of its geometric and electronic structures with phosphoenol pyruvate,Eq. 12 [28]. 

Occasionally, one can maintain initial rate conditions by using a coupled reaction system to regenerate one of the limiting substrates. For example, to regenerate ATP in a phosphotransferase reaction, one can use creatine phosphate and creatine kinase acetylphosphate and acetate kinase or phosphoenolpyruvate and pyruvate kinase. 

FIGURE 13-3 Hydrolysis of phosphoenolpyruvate (PB3). Catalyzed by pyruvate kinase, this reaction is followed by spontaneous tautomerization of the product, pyruvate. Tautomerization is not possible in PER and thus the products of hydrolysis are stabilized relative to the reactants Fiesonance stabilization of P, also occurs, as shown in Figure 13-1. 

Transfer of the Phosphoryl Group from Phosphoenolpyruvate to ADP The last step in glycolysis is the transfer of the phosphoryl group from phosphoenolpyruvate to ADP, catalyzed by pyruvate kinase, which requires K+ and either Mg2+ or Mn2+ ... 

The first "roadblock" to overcome in the synthesis of glucose from pyruvate is the irreversible conversion in glycolysis of pyruvate to phosphoenolpyruvate (PEP) by pyruvate kinase. In gluconeogenesis, pyruvate is first carboxylated by pyruvate carboxylase to oxaloacetate (OAA), which is then converted to PEP by the action of PEP-carboxykinase (Figure 10.3). 

In this one-pot procedure NeuAc 16 is generated from ManNAc 15 and pyruvic acid in situ with sialic acid aldolase and then converted irreversibly to CMP-NeuAc 17. CMP is converted to CDP with myokinase and ATP. The released ADP is converted to ATP with pyruvate kinase and PEP. CDP is then converted to CTP also with pyruvate kinase and phosphoenolpyruvate (PEP). The formed CTP reacts with NeuAc catalyzed by NeuAc synthetase to give 17. 

An example is illustrated in Fig. 12-20. In this experiment685 the relative areas of the 31P signals of ADP (one for free ADP and one, slightly more intense, for MgADP) and of the signal for phosphoenolpyruvate (PEP) were measured in the absence of enzyme and in the presence of a catalytic amount of pyruvate kinase (Fig. 12-20A). The results verified that the equilibrium constant for the overall reaction (Eq. 12-33) is very high (3300). 

The 500-residue subunits of pyruvate kinase consist of four domains,891 the largest of which contains an 8-stranded barrel similar to that present in triose phosphate isomerase (Fig. 2-28). Although these two enzymes catalyze different types of reactions, a common feature is an enolic intermediate. One could imagine that pyruvate kinase protonates its substrate phosphoenolpyruvate (PEP) synchronously with the phospho group transfer (Eq. 12-42). However, the enzyme catalyzes the rapid conversion of the enolic form of pyruvate to the oxo form (Eq. 12-43) adding the proton sterospecifically to the si face. This and other evidence favors the enol as a true intermediate... 

Figure 10.4 The abolition of positive cooperativity on the binding of allosteric effectors to some enzymes. Note the dramatic increases in activity at low substrate concentrations on the addition of adenosine monophosphate to isocitrate dehydrogenase, of deoxycytosine diphosphate to deoxythymidine kinase, and of fructose 1,6-diphosphate to pyruvate kinase this shows how the activity may be switched on by an allosteric effector (PEP = phosphoenolpyruvate). [From J. A. Hathaway and D. E. Atkinson, J. Biol. Chem. 238,2875 (1963) R. Okazaki and A. Kornbcrg, J. Biol. Chem. 239,275 (1964) R. Haeckel, B. Hess, W. Lauterhom, and K.-H. Wurster, Hoppe-Seyler s Z. Physiol. Chem. 349, 699 (1968).]...

The final energy payoff in the glycolytic pathway occurs in the hydrolysis of phosphoenolpyruvate to pyruvate and the concomitant phosphorylation of ADP to ATP. Two molecules of ATP are produced for each molecule of hexose phosphate consumed, bringing the net yield of ATP to two molecules for each molecule of glucose (two molecules of ATP are regenerated in the phosphoglycerate kinase step and two in this step, and two are consumed in the hexoki-nase and phosphofructokinase steps). 

The first step in the gluconeogenic direction involves the formation of phosphoenolpyruvate from pyruvate. Reversal of the pyruvate kinase reaction requires at least two ATP-to-ADP conversions. One means by which this is done is shown in figure 12.26. 

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