Properties of Cys21-mutated muscle acylphosphatases.
Cys21 is an invariant residue in muscle acylphosphatases, but is absent in the erythrocyte isozymes. To assess the importance of this residue in the muscle isozymes for catalytic, structural, and stability properties, two gene mutants have been prepared by oligonucleotide-directed mutagenesis and expressed in Escherichia coli cells; in these mutants, the codon for Cys21 was replaced by those for Ser and Ala, respectively. The two mutant enzymes, purified by immunoaffinity chromatography, showed kinetic and structural properties similar to those of the wild-type recombinant enzyme; however, the specific activity of the two mutants, especially that of the C21A mutant, was lower. The urea and thermal stabilities of the mutant enzymes were reduced with respect to those of the wild-type form, contrary to the susceptibility to inactivation by mercuric ions. The reported data support the possibility that Cys21 is involved in the stabilization of the enzyme active-site conformation.
Modesti
A
A
Department of Biochemical Sciences, University of Florence, Italy.
Taddei
N
N
Chiti
F
F
Bucciantini
M
M
Magherini
F
F
Rigacci
S
S
Stefani
M
M
Raugei
G
G
Ramponi
G
G
D017766:Acid Anhydride Hydrolases / Q000378:metabolism*; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D017931:DNA Primers / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression / Q000235:genetics; D006736:Horses / Q000378:metabolism; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008628:Mercury / Q000494:pharmacology; D008969:Molecular Sequence Data; D009132:Muscles / Q000201:enzymology; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D017354:Point Mutation / Q000235:genetics; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014508:Urea / Q000494:pharmacology; D000097024:Acylphosphatase
D017766:Acid Anhydride Hydrolases; D003545:Cysteine; D017931:DNA Primers; D008628:Mercury; D011994:Recombinant Proteins; D014508:Urea; D000097024:Acylphosphatase