Kinetics of the activation of rat liver pyruvate kinase by fructose 1,6-disphosphate and methods for characterizing hysteretic transitions

Abstract

1. Kinetics of fructose 1,6-diphosphate activation of liver pyruvate kinase type I inhibited with MgATP and l-alanine are described as a function of enzyme and fructose 1,6-diphosphate concentrations. These results can be explained by a single pseudo-first-order transition of the enzyme into an active form, independent of the enzyme concentration. This rate constant, kapp.=0.24s-1 with 0.02mm-fructose 1,6-diphosphate (t0.9 ≃ 10s where t0.9 is the time for 90% conversion), is an increasing function of fructose 1,6-diphosphate concentration far beyond that needed to maximally activate enzyme equilibrated with fructose 1,6-diphosphate (about 20μm). 2. The model equations are best analysed with numerical techniques which are described. These techniques are useful in studying similar slow transients frequently observed in stopped-flow studies of enzymes. 3. Shorter transients (t0.9=0.5–1.5s) were observed in the kinetic response of the enzyme to the addition of MgATP or phosphoenolpyruvate, but were not further characterized.