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 Km and Fmax 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 Km and Fmax 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.
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