GENETICALLY-ENGINEERED ZINC-CHELATING ADENYLATE KINASE FROM ESCHERICHIA-COLI WITH ENHANCED THERMAL-STABILITY

In contrast with adenylate kinase from Gram-negative bacteria, the enz yme from Gram-positive organisms harbors a structural Zn2+ bound to 3 or 4 Cys residues in the structural motif Cys-X-2-Cys-X-16-Cys-X-2-Cys /Asp. Site-directed mutagenesis of His(126), Ser(129), Asp(146), and T hr(149) (corresponding to Cys(130), Cys(133), Cys(150), and Cys(153) i n adenylate kinase from Bacillus stearothermophilus) in Escherichia co li adenylate kinase was undertaken for determining whether the presenc e of Cys residues is the only prerequisite to bind zinc or (possible) other cations. A number of variants of adenylate kinase from E. coli, containing 1-4 Cys residues were obtained, purified, and analyzed for metal content, structural integrity, activity, and thermodynamic stabi lity. All mutants bearing 3 or 4 cysteine residues acquired zinc bindi ng properties. Moreover, the quadruple mutant exhibited a remarkably h igh thermal stability as compared with the wild-type form with preserv ation of the kinetic parameters of the parent enzyme.