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J Biol Chem, Vol. 273, Issue 30, 19097-19101, July 24, 1998
From the In contrast with adenylate kinase from
Gram-negative bacteria, the enzyme from Gram-positive organisms harbors
a structural Zn2+ bound to 3 or 4 Cys residues in the
structural motif
Cys-X2-Cys-X16-Cys-X2-Cys/Asp. Site-directed mutagenesis of His126, Ser129,
Asp146, and Thr149 (corresponding to
Cys130, Cys133, Cys150, and
Cys153 in adenylate kinase from Bacillus
stearothermophilus) in Escherichia coli adenylate
kinase was undertaken for determining whether the presence 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 stability. All mutants bearing 3 or 4 cysteine residues
acquired zinc binding properties. Moreover, the quadruple mutant
exhibited a remarkably high thermal stability as compared with the
wild-type form with preservation of the kinetic parameters of the
parent enzyme.
Genetically Engineered Zinc-chelating Adenylate Kinase from
Escherichia coli with Enhanced Thermal Stability
,
, and
Laboratoire de Chimie Structurale des
Macromolécules, Institut Pasteur, 75724 Paris Cedex 15, France,
§ INSERM U 350, Institut Curie-Recherche, 91405 Orsay Cedex,
France, ¶ Laboratoire de Sciences des Sols et Hydrologie, Institut
National d'Agronomie, Centre de Grignon, 78850 Thiverval, France, and
Department of Ophthalmology, University
of Missouri, Columbia, Missouri 65212
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
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