title: [Investigation of active center of deoxynucleoside monophosphate kinase of bacteriophage T5 by site-directed mutagenesis].
abstract: Based on the computer model of active center of bacteriophage T5 deoxyribonucleoside monophosphate kinase amino acid residues essential for the enzyme activity were determined. As the result of site-directed mutagenesis, cloning and expression of the gene in E. coli series of proteins were obtained with single amino acid substitutions of conservative active center residues--S13A, D16N, T17N, T17S, R130K, K131E, Q134A, G137A, T138A, W150F, W150A, D170N, R172I, E176Q. Electrophoretically homogeneous preparations of mutant forms were purified using ion exchange and affinity chromatographic steps. Measuring of the specific enzyme activities of these enzymes for the natural acceptors of phosphoryl group (dAMP, dCMP, dGMP, dTMP) revealed that substitutions of charged residues of NMP-binding domain-namely, R130, R172, D170 and E176-lead to almost complete loss of enzyme activity. It was shown that presence of OH-group at position 17 is also important for catalytic activity. Based on the changes in specific activities we suppose that arginine residues at positions 130 and 172 participate in binding of γ-phosphoryl of donor and α-phosphoryl of acceptor. Also, aspartic acid at 16 position of ATP-binding site (P-loop) probably assists in the binding of acceptor, first of all dTMP. Unequal decrease in enzyme activities for different substrates of partially active mutants--G137A, T138A, T17N, Q134A, S13A, D16N--indicate that in the binding of various substrates different amino acid residues take part.
authors.forename: G V
authors.initials: GV
authors.lastname: Mikulinskaia
authors.forename: S A
authors.initials: SA
authors.lastname: Taran
authors.forename: Iu S
authors.initials: IuS
authors.lastname: Skoblov
authors.forename: S A
authors.initials: SA
authors.lastname: Feofanov
mesh_terms: D000595:Amino Acid Sequence; D001435:Bacteriophages / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D017856:Phosphotransferases (Phosphate Group Acceptor) / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification
chemical_list: D017856:Phosphotransferases (Phosphate Group Acceptor); C020015:(deoxy)nucleoside-phosphate kinase