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 exchange between nucleoside triphosphates and nucleoside diphosphates. Its 17 kDa subunits are highly conserved throughout evolution in both sequence and tertiary structure. Using site-directed mutagenesis we investigated the function of 8 amino acids (Lys16, Tyr56, Arg92, Thr98, Arg109, Asn119, Ser124, and Glu133) that are totally conserved among all nucleoside diphosphate kinases known to date. The mutant proteins all show decreased specific activity and support roles for these residues in catalysis, substrate binding, or both, as was previously proposed on the basis of the x-ray structure (Moréra, S., Lascu, I., Dumas, C., LeBras, G., Briozzo, P., Véron, M., and Janin, J. (1994) Biochemistry 33, 459-467). Furthermore, residues Lys16, Arg109, and Asn 119 were identified to play important roles in conformational stability or subunit interactions. We show that Lys16 and Asn119 form a rigid structure that is important for enzymatic function and that Arg109, known to interact with the phosphate moiety of the substrate, also plays an important role in subunit association. The dual roles of Lys16, Arg109, and Asn119 in both substrate binding and subunit assembly provide further evidence for a functional coupling between catalytic activity and quaternary structure in nucleoside diphosphate kinase.
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Unité de Biochimie Cellulaire, CNRS-URA 1129, Institut Pasteur, Paris, France.
Dammann
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Bominaar
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Véron
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D000595:Amino Acid Sequence; D000596:Amino Acids / Q000235:genetics; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D004023:Dictyostelium / Q000201:enzymology; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009701:Nucleoside-Diphosphate Kinase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides; D017434:Protein Structure, Tertiary
D000596:Amino Acids; D009838:Oligodeoxyribonucleotides; D009701:Nucleoside-Diphosphate Kinase