Isotopic exchange studies on the type-L pyruvate kinase from pig liver.

Abstract

The glycolytic enzyme pyruvate kinase (EC 2.7.1.40) is known to occur in several isoenzyme forms in mammalian tissues (Tanaka et al., 1967). The phosphoryl transfer from phosphoenolpyruvate to ADP catalysed by the type-hl rabbit muscle enzyme has been shown to occur by a direct transfer (sequential) mechanism and not via a stable phosphorylated-enzyme intermediate (non-sequential or Ping Pong mechanism) (Reynard et al., 1961). The type-L enzyme from liver shows a sigmoidal relationship between initial velocity and phosphoenolpyruvate concentration unless activated by fructose 1,6-bisphosphate, when a hyperbolic relationship is obtained together with a lowering of the (phosphoenolpyruvate) value. The mechanism of phosphoryl transfer in the type-L enzyme is less certain. Results of an initial-rate kinetic analysis of the pig liver enzyme have been interpreted as implying that a non-sequential mechanism operates (MacFarlane & Ainsworth, 1974). Kinetic analysis of the enzymes from human liver (Balinsky et al., 1973) and from the hepatopancreas of Carcinus maenas (Giles et al., 1975), however, indicate a sequential mechanism. If pyruvate kinase catalyses a reaction involving the formation of a stable phosphorylated-enzyme intermediate, isotopic exchange between ADP and ATP (or phosphoenolpyruvate and pyruvate) should occur as illustrated by the following partial reactions E+ATP + (E*ATP + E.P*ADP) + EsPSADP