Phosphoenolpyruvate pyruvate conversion

The activity of the ATP-forming enzyme complex V is usually assessed by determining the reverse reaction ATP — ADP + Pi. The reaction is coupled to reactions catalyzed by pyruvate kinase (ADP + phosphoenolpyruvate —> pyruvate + ATP) and lactate dehydrogenase (pyruvate + NADH — lactate -F NAD+). This final reaction can be followed spectrophotometrically by measuring NADH at 340 nm. The activity of complex V (ATPase) can be derived from the rate of NADH conversion in the presence and absence of the specific complex V inhibitor oligomycin. 

As these freely reversible aldol additions often have less favorable equilibrium constants [30,34], synthetic reactions usually have to be driven by an excess of pyruvate to achieve satisfactory conversions. A few related enzymes have been identified that utilize phosphoenolpyruvate instead of pyruvate, which upon C—C bond formation releases inorganic phosphate, and thus renders the aldol addition essentially irreversible (Figure 10.4) [16]. Although attractive from a synthetic point ofview, the latter enzymes have been less studied as yet for preparative applications [35]. 

Figure 5.13 Conversion of pyruvate to oxaloacetate and then to phosphoenolpyruvate.

Following the conversion of pyruvate into oxaloacetate, phosphoenolpyruvate carboxykinase (PEP CK) catalyses a decarboxylation to form PEP (Figure 6.42). This... 

Figure 6-7. Conversion of mitochondrial pyruvate to cytosolic phosphoenolpyruvate to initiate gluconeogenesis. Oxaloacetate cannot pass across the inner mitochondrial membrane, so it is reduced to malate, which can do so.

The overall reaction has a large, negative standard free-energy change, due in large part to the spontaneous conversion of the enol form of pyruvate to the keto form (see Fig. 13-3). The AG ° of phosphoenolpyruvate... 

Conversion of Pyruvate to Phosphoenolpyruvate Requires Two Exergonic Reactions... 

Vertebrates cannot convert fatty acids, or the acetate derived from them, to carbohydrates. Conversion of phosphoenolpyruvate to pyruvate (p. 532) and of pyruvate to acetyl-CoA (Fig. 16-2) are so exergonic as to be essentially irreversible. If a cell cannot convert acetate into phosphoenolpyruvate, acetate cannot serve as the starting material for the gluconeogenic pathway, which leads from phosphoenolpyruvate to glucose (see Fig. 15-15). Without this capacity, then, a cell or organism is unable to convert fuels or metabolites that are degraded to acetate (fatty acids and certain amino acids) into carbohydrates. 

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). 

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... 

Gluconeogenesis Consumes ATP Conversion of Pyruvate to Phosphoenolpyruvate Requires Two High Energy Phosphates Conversion of Phosphoenolpyruvate to Fructose-1,6-bisphosphate Uses the Same Enzymes as Glycolysis... 

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. 

The carboxylation of pyruvate supplies a significant portion of the thermodynamic push for the next step in the sequence. This is because the free energy change for decarboxylation of /3-keto carboxylic acids such as oxaloacetate is large and negative. The oxaloacetate formed from pyruvate by carboxylation is converted to phosphoenolpyruvate in a reaction catalyzed by phosphoenolpyruvate carboxyki-nase. In many species, including mammals, this reaction involves a GTP-to-GDP conversion. 

If we add the equations for the reactions catalyzed by pyruvate carboxylase, phosphoenolpyruvate carboxykinase, and nucleoside diphosphate kinase, we obtain the overall reaction for conversion of pyruvate to phosphoenolpyruvate. 

Glycolysis provides the main source of ATP in Trypanosoma brucei, E histolytica, and G lamblia, which possess pyruvate kinase as well as a pyruvate phosphate dikinase for converting phosphoenolpyruvate (PEP) to pyruvate and generating ATP. Pyruvate phosphate dikinase is not a homolog of pyruvate kinase but is closely related to PEP synthase from bacteria. The enzyme catalyzes conversion of PEP to pyruvate accompanied by the synthesis of ATP from AMP and pyrophosphate. Genes encoding the enzyme have been isolated from E histolytica and G lamblia and have demonstrated considerable structural divergences. No specific inhibitor of this enzyme has yet been identified. 

Pathway of C02 in Gluconeogenesis In the first bypass step of gluconeogenesis, the conversion of pyruvate to phosphoenolpyruvate (PEP), pyruvate is carboxylated by pyruvate carboxylase to oxaloacetate, which is subsequently decarboxylated to PEP by PEP carboxykinase (Chapter 14). Because the addition of C02 is directly followed by the loss of C02, you might expect that in tracer experiments, the 14C of 14C02 would not be incorporated into PEP, glucose, or any intermediates in gluconeogenesis. 

Step 10, the final step in glycolysis, is the irreversible conversion of phosphoenolpyruvate to pyruvate, catalyzed by pyruvate kinase. This is the second energy-yielding step in the glycolytic pathway, and produces ATP Mg2+ is required here, too. 

Step A, the conversion of pyruvate to phosphoenolpyruvate, is accomplished by a circuitous process commencing with pyruvate entering the mitochondrion, which for gluconeogenesis to occur must be in a high-energy state. Under these conditions, the mitochondrial enzyme pyruvate carboxylase catalyzes the conversion of pyruvate to oxaloacetate-. 

The Conversion of Pyruvate into Phosphoenolpyruvate Begins with the Formation of Oxaloaeetate... 

A third fate of pyruvate is its carboxylation to oxaloacetate inside mitochondria, the first step in gluconeogenesis. This reaction and the subsequent conversion of oxaloacetate into phosphoenolpyruvate bypass an irreversible step of glycolysis and hence enable glucose to be synthesized from pyruvate. The carboxylation of pyruvate is also important for replenishing intermediates of the citric acid cycle. Acetyl CoA activates pyruvate carboxylase, enhancing the synthesis of oxaloacetate, when the citric acid cycle is slowed by a paucity of this intermediate. 

There will be no labeled carbons. The CO2 added to pyruvate (formed from the lactate) to form oxaloacetate is lost with the conversion of oxaloacetate into phosphoenolpyruvate. 

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