Mechanism of phosphoryl transfer by nucleoside diphosphate kinase - pH dependence and role of the active site Lys16 and Tyr56 residues

Nucleoside diphosphate (NDP) kinase phosphorylates nucleoside diphosphates with little specificity for the base and the sugar. Although nucleotide ana logues used in antiviral therapies are also metabolized to their triphospha te form by NDP kinase, their lack of the 3'-hydroxyl of the ribose, which a llows them to be DNA chain terminators, severely impairs the catalytic effi ciency of NDP kinase. We have analyzed the kinetics parameters of several m utant NDP kinases modified on residues (Lys16, Tyr56, Asn119) interacting w ith the gamma -phosphate and/or the 3'-OH of the Mg2+-ATP substrate. We com pared the relative contributions of the active-site residues and the substr ate 3'-OH for point mutations on Lys16, Tyr56 and Asn119. Analysis of addit ional data from pH profiles identify the ionization state of these residues in the enzyme active form. X-ray structure of K16A mutant NDP kinase shows no detectable rearrangement of the residues of the active site.