Phosphopyruvate kinase

ITP is the only nucleotide that is known to substitute for GTP. The reaction was thought for many years to use ATP, but the apparent utilization of ATP was the result of the presence of other nucleotides in ATP preparations and the presence of nucleoside diphosphate kinase in the enzyme preparations. Thus the active nucleotide reacted in catalytic quantities and the nucleoside diphosphate produced accepted the terminal phosphate of ATP to give the appearance of participation of ATP in the decarboxylation of oxalacetate. Another complication in the analysis of this reaction is the secondary reaction of phosphopyruvate with nucleoside diphosphates. Phosphopyruvate kinase, which is present in crude extracts, transfers phosphate from the product of decarboxylation to the nucleotide also formed in the reaction, so that no phosphate transfer is seen, and the reaction appears to be a simple decarboxylation. It... 

Phosphate is again present in an energy-rich form (namely the enol ester). It can be transferred by phosphopyruvate kinase to ADP this transfer affords pyruvic acid, which is the most important metabolite of both anaerobic and aerobic carbohydrate metabolism. 

Sodium fluoride (104) (1-10 mM) inhibits two enzymes of glycolysis the enolase (phosphopyruvate hydratase) and pyruvate kinase. Therefore, aerobic glucose utilization and lactate formation are blocked. 

In the preceding sections the conversion of purines and purine nucleosides to purine nucleoside monophosphates has been discussed. The monophosphates of adenosine and guanosine must be converted to their di- and triphosphates for polymerization to RNA, for reduction to 2 -deoxyribonucleoside diphosphates, and for the many other reactions in which they take part. Adenosine triphosphate is produced by oxidative phosphorylation and by transfer of phosphate from 1,3-diphosphoglycerate and phosphopyruvate to adenosine diphosphate. A series of transphosphorylations distributes phosphate from adenosine triphosphate to all of the other nucleotides. Two classes of enzymes, termed nucleoside mono-phosphokinases and nucleoside diphosphokinases, catalyse the formation of the nucleoside di- and triphosphates by the transfer of the terminal phosphoryl group from adenosine triphosphate. Muscle adenylate kinase (myokinase)... 

Pyruvate kinase, phosphopyruvate kbtase (EC 2.7.1.40) a widely distributed, metal-ion dependent phosphotransferase, present in yeast, muscle, liver, erythrocytes and other organs and cells. It catalyses the last reaction of glycolysis Phosphoeno/pyruvate (PEP) + ADP -> Pyruvate + ATP (substrate level phosphorylation). Each subunit of P. k. forms an intermediate, cyclic, ternary metal bridge complex ... 

A reversible reaction catalyzes the conversion of pyruvate to phosphopyruvate, and the enzyme involved is pyruvic kinase. The equilibrium of that reaction is on the side of the formation of ATP. Thus, pyruvate kinase is the enzyme responsible for the conversion of phosphoenolpyruvate to pyruvate. The enzyme has been crystallized from muscle it requires ADP, potassium, and magnesium and is noncompetitively inhibited by some estrogenic steroids. Steroids alter the enzyme s viscosity and electrophoretic properties. From this observation, it was assumed that steroids act by modifying the protein molecule. 

Polynucleotide phosphorylase can be assayed in several ways. The liberation of inorganic phosphate can be used to follow the reaction. Both disappearance of acid-soluble nucleotides and formation of acid-insoluble nucleotides have been measured. The reverse reaction in the presence of P Mabeled phosphate allows an exchange reaction, the incorporation of P into nucleotides, to be used as an assay. The exchange reaction was the first reaction of this enzyme to be discovered, and studies on the responsible enzyme led to the finding of polynucleotide synthesis. Another assay based on the reverse reaction has been devised for rapid spectrophoto-metric determinations. A polymer of adenylic acid is incubated with the phosphorylase in the presence of phosphate, phosphopyruvate, pyruvate kinase, DPNH, and lactic dehydrogenase. The formation of ADP is thus coupled with the formation of pyruvate which reacts stoichiometrically with PPNH, so that the entire reaction can be followed at 340 m/a. 

Fig. 15.2 Pyruvate metabolism, gluconeogenesis and glycolysis. Key enzymes shown are 1, fructose 1-phosphate aldolase (EC 4.1.2.13) 2, fructose 1,6-bisphosphate aldolase (EC 4.1.2.13) 3, NAD rglyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.12) 4, 3-phosphoglycerate kinase (EC 2.T.2.3) 5, phosphoglyceromutase (EC 2.7.5.3) 6, phosphopyruvate hydratase (enolase) (EC 4.2.1.11) 7, pyruvate kinase (EC 2.7.1.40) 8, pyruvate carboxylase (EC 6.4.1.1) 9, phosphopyruvate carboxylase...

---