Phosphoenolpyruvate Carboxykinase from Bakers' Yeast

• Published in: The Journal of biological chemistry 1974-06, Vol.249 (11), p.3356-3365
• Main Authors: Cannata, Joaquin J.B., de Flombaum, Maria A.C.
• Format: Article
• Language: English
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(19)42580-5

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 Mn 2+ 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 Mn 2+ as cation is analyzed in Lineweaver-Burk double reciprocal form a biphasic plot, indicative of activation either by Mn-ATP 2- or ATP 4- , is obtained. Excess of ATP over MnCl 2 is inhibitory. Regarding cation requirement, Mn 2+ is the most effective at pH 8.1. On the other hand, at pH 5.9 Mn 2+ and Cd 2+ are equally effective. At pH 8.1 the curve for rate plotted against MnCl 2 concentration is sigmoidal, and maximal activity is always reached at a 1:1 ratio between ATP and MnCl 2 concentrations. Excess of MnCl 2 over ATP is inhibitory. Addition of low concentrations of CdCl 2 ( < 10 -2 m m ) shifts the sigmoidal shaped curves to hyperbolic ones. When the ratio of MnCl 2 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 CdCl 2 . At pH 6.7 and 5.9 the kinetics of MnCl 2 activation is hyperbolic. Addition of CdCl 2 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 Mn 2+ and Cd +2 can fulfill the free divalent cation requirement.