INVESTIGATION OF THE ACTIVE-SITE AND THE CONFORMATIONAL STABILITY OF NUCLEOSIDE DIPHOSPHATE KINASE BY SITE-DIRECTED MUTAGENESIS

Nucleoside-diphosphate kinase (EC 2.7.4.6) catalyzes phosphate exchang e between nucleoside triphosphates and nucleoside diphosphates. Its 17 kDa subunits are highly conserved throughout evolution in both sequen ce and tertiary structure. Using site-directed mutagenesis we investig ated the function of 8 amino acids (LyS(16), Tyr(56), Arg(92), Thr(98) , Arg(109), Asn(119), Ser(124) and Glu(193)) that are totally conserve d among all nucleoside diphosphate kinases known to date. The mutant p roteins all show decreased specific activity and support roles for the se residues in catalysis, substrate binding, or both, as was previousl y proposed on the basis of the x-ray structure (Morera, S., Lascu, I., Dumas, C., LeBras, G., Briozzo, P., Veron, M., and Janin, J. (1994) B iochemistry 33, 459-467), Furthermore, residues Lys(16), Arg(109), and Asn(119) were identified to play important roles in conformational st ability or subunit interactions. We show that Lys(16) and Asn(119) for m a rigid structure that is important for enzymatic function and that Arg(109), known to interact with the phosphate moiety of the substrate , also plays an important role in subunit association. The dual roles of Lys(16), Arg(109), and Asn(119) in both substrate binding and subun it assembly provide further evidence for a functional coupling between catalytic activity and quaternary structure in nucleoside diphosphate kinase.