The kinetics of rabbit muscle pyruvate kinase. Initial-velocity, substrate- and product-inhibition and isotopic-exchange studies of the reverse reaction.
1. An assay, based on the transfer of label from [gamma-32P]ATP to [32P]phosphoenolpyruvate, suitable for a steady-state kinetic analysis of pyruvate kinase in the reverse direction (i.e. phosphoenolpyruvate synthesis), is described. 2. This assay was used in a kinetic investigation of the rabbit muscle enzyme including initial-rate and product-inhibition experiments, at a pH of 7.4 and constant concentrations of total K+ and free Mg2+. 3. These studies indicate that there is a random release of ADP and phosphoenolpyruvate from the enzyme and that there is a competitive substrate inhibition by ATP. Some of the results were suggestive that the rapid-equilibrium assumption, generally used for this enzyme was not valid. 4. Techniques were developed to measure the rate of isotopic exchange between all the substrate-product pairs. 5. By using these techniques the rates of isotopic exchange at chemical equilibrium were measured. The results indicate that this enzyme does not catalyse a truly rapid-equilibrium random mechanism, although in the forward reaction all initial-rate data obtained to date are consistent with this assumption.
D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate / Q000302:isolation & purification; D000818:Animals; D007700:Kinetics; D008274:Magnesium; D009132:Muscles / Q000201:enzymology*; D010728:Phosphoenolpyruvate / Q000378:metabolism; D010761:Phosphorus Radioisotopes; D011770:Pyruvate Kinase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011817:Rabbits
D010761:Phosphorus Radioisotopes; D000244:Adenosine Diphosphate; D010728:Phosphoenolpyruvate; D000255:Adenosine Triphosphate; D011770:Pyruvate Kinase; D008274:Magnesium