A Kinetic Study of Nucleotide Interactions with Pyruvate Kinase

The apparent broad specificity of the enzyme pyruvate kinase for the nucleoside diphosphate substrate has been re-examined. Nucleoside diphosphokinase activity was found to be present but was active only when exogenous adenosine triphosphate was present. The apparent K_m and F_max values were determined at several pH values between 6.0 and 9.0 for a number of nucleoside diphosphates. A doubly ionized phosphoryl group on the nucleotides and/or an imidazole group on the enzyme appear to be essential for nucleotide binding. Both the apparent K_m and F_max values decrease between pH 7.5 and 9.0, which suggests the involvement of an ionized α-amino group in the reaction. The results of the determinations of kinetic parameters in the pH range of 7-8 are discussed in relation to a proposed mechanism in which the nucleoside diphosphate is bound to the enzyme on two regions in an obligatory order, with the β-phosphoryl group binding first and the nucleoside portion binding second.