Investigation of the active site and the conformational stability of nucleoside diphosphate kinase by site-directed mutagenesis.

摘要: Abstract Nucleoside-diphosphate kinase (EC 2.7.4.6) catalyzes phosphate exchange between nucleoside triphosphates and diphosphates. Its 17 kDa subunits are highly conserved throughout evolution in both sequence tertiary structure. Using site-directed mutagenesis we investigated the function of 8 amino acids (Lys16, Tyr56, Arg92, Thr98, Arg109, Asn119, Ser124, Glu133) that totally among all diphosphate kinases known to date. The mutant proteins show decreased specific activity support roles for these residues catalysis, substrate binding, or both, as was previously proposed on basis x-ray structure (Morera, S., Lascu, I., Dumas, C., LeBras, G., Briozzo, P., Veron, M., Janin, J. (1994) Biochemistry 33, 459-467). Furthermore, Lys16, Asn 119 were identified play important conformational stability subunit interactions. We Lys16 Asn119 form a rigid is enzymatic interact with moiety substrate, also plays an role association. dual binding assembly provide further evidence functional coupling catalytic quaternary kinase.