An investigation of the interactions of the allosteric modifiers of pyruvate kinase with the enzyme from Carcinus maenas hepatopancreas

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Abstract
Pyruvate kinase [EC 2.7.1.40] purified from the hepatopancreas of C. maenas exhibited sigmoidal saturation kinetics with respect to the substrate phosphoenolpyruvate in the absence of the allosteric activator fructose 1,6-bisphosphate, but normal hyperbolic saturation was seen in the presence of this activator. The activation appears to be the result of a decrease in the s0.5 (phosphoenolpyruvate) and not to a change in Vmax. In the presence of ADP and ATP at a constant nucleotide-pool size the results indicate that phosphoenolpyruvate co-operativity is lost on increasing the [ATP]/[ADP] ratio. Paralleling this change is the observation that the fructose 1,6-bisphosphate activation became less as the [ATP]/[ADP] ratio was increased. This was due to the enzyme exhibiting a near-maximal activity in the absence of activator. L-Alanine inhibited the enzyme, but homotropic co-operative interactions were only seen with a cruder (100,000 g supernatant) enzyme preparation. The inhibition by alanine could be overcome by increasing the concentration of either phosphoenolpyruvate or fructose 1,6-bisphosphate, although increasing the L-alanine concentration did not appear to be able to reverse the activation by fructose 1,6-bisphosphate. In the presence of a low concentration of phosphoenolpyruvate, increasing the concentration of the product, ATP, caused and initial increase in enzyme activity, followed by an inhibitory phase. In the presence of either fructose 1,6-bisphosphate or L-alanine only inhibition was seen. The inhibition by ATP could not be completely reversed by fructose 1,6-bisphosphate.