Phosphoenolpyruvate Carboxykinase from Bakers' Yeast: KINETICS OF PHOSPHOENOLPYRUVATE FORMATION

Abstract

Abstract The kinetic data of phosphoenolpyruvate formation catalyzed by crystalline bakers' yeast P-enolpyruvate carboxykinase were examined with regard to nucleotide and divalent cation absolute requirements. With Mn2+ as cation, the pH profile shows a peak at pH 8.3 with a shoulder in the vicinity of pH 6. There is no evidence to indicate that this is due to the presence of a second enzyme. The enzyme shows a rather high degree of specificity towards ATP (or deoxyadenosine triphosphate). When initial rate data from ATP saturation curve with Mn2+ as cation is analyzed in Lineweaver-Burk double reciprocal form a biphasic plot, indicative of activation either by Mn-ATP2- or ATP4-, is obtained. Excess of ATP over MnCl2 is inhibitory. Regarding cation requirement, Mn2+ is the most effective at pH 8.1. On the other hand, at pH 5.9 Mn2+ and Cd2+ are equally effective. At pH 8.1 the curve for rate plotted against MnCl2 concentration is sigmoidal, and maximal activity is always reached at a 1:1 ratio between ATP and MnCl2 concentrations. Excess of MnCl2 over ATP is inhibitory. Addition of low concentrations of CdCl2 ( l 10-2 mm) shifts the sigmoidal shaped curves to hyperbolic ones. When the ratio of MnCl2 and ATP concentrations is kept constant and equal to one, the plot of rate versus cofactors concentration is hyperbolic both in the presence and absence of CdCl2. At pH 6.7 and 5.9 the kinetics of MnCl2 activation is hyperbolic. Addition of CdCl2 enhances enzyme activity greatly also at those pH values. From these results it is proposed that at least two binding sites, related to cation requirement, are present on the yeast enzyme, one binding a cation-nucleotide complex, the true substrate, and the other a free divalent cation. Binding of the free cation at the latter site greatly enhances the affinity of the former for the cation-nucleotide substrate. Apparently only Mn2+ and Cd+2 can fulfill the free divalent cation requirement.