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.
The kinetics of rabbit muscle pyruvate kinase. Initial-velocity, substrate- and product-inhibition and isotopic-exchange studies of the reverse reaction
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I G Giles, P C Poat, K A Munday; The kinetics of rabbit muscle pyruvate kinase. Initial-velocity, substrate- and product-inhibition and isotopic-exchange studies of the reverse reaction. Biochem J 1 September 1976; 157 (3): 577–589. doi: https://doi.org/10.1042/bj1570577
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