The effect of a varying Mg2+free concentration upon the kinetic behaviour of human liver L-type pyruvate kinase

Abstract

We have proposed negative cooperativity for human liver L-type pyruvate kinase, a phenomenon also observed in: Lineweaver-Burk plots V⁻¹ versus [phosphoenolpyruvatel]⁻¹ were straight lines at low phosphoenolpyruvate concentrations, and bent downward near they axis (high phosphoenolpyruvate concentrations). Our conclusions were drawn from experiments performed at Mg²⁺total = 12 mM or 20 mM. However, phosphoenolpyruvate has been proposed to bind Mg²⁺free with a Keq = 5.5 X 1O⁻³ M⁻¹ at 25°C. So, at high phosphoenolpyruvate concentrations Mg²⁺free would be removed from the reaction mixture. We investigated the hypothesis that the phenomenon interpreted as negative cooperativity is in fact due to removing inhibitory Mg²⁺free from the reaction mixture at high phosphoenolpyruvate concentrations. Our results are in favour of this hypothesis.