TY - JOUR
T1 - Arginine-23 is involved in the catalytic site of muscle acylphosphatase
AU - Taddei, Niccolò
AU - Stefani, Massimo
AU - Vecchi, Manuela
AU - Modesti, Alessandra
AU - Raugei, Giovanni
AU - Bucciantini, Monica
AU - Magherini, Francesca
AU - Ramponi, Giampietro
PY - 1994/9/21
Y1 - 1994/9/21
N2 - Three mutants of human muscle acylphosphatase in which arginine-23 was replaced by glutamine, histidine and lysine, respectively, were prepared by oligonucleotide-directed mutagenesis of a synthetic gene coding for the enzyme. All mutants, purified by affinity chromatography, were almost completely unable to catalyze the hydrolysis of the substrate. 1H-NMR spectroscopy experiments showed the absence of any major conformational changes of the three mutants with respect to the wild-type recombinant enzyme. Equilibrium dialysis experiments demonstrated that the mutated proteins lost the ability of binding inorganic phosphate, a competitive inhibitor of the enzyme. These results strongly support an involvement of arginine-23 at the phosphate binding-site of acylphosphatase, confirming the hypothesis of the existence of a phosphate binding structural motif recently proposed by other authors.
AB - Three mutants of human muscle acylphosphatase in which arginine-23 was replaced by glutamine, histidine and lysine, respectively, were prepared by oligonucleotide-directed mutagenesis of a synthetic gene coding for the enzyme. All mutants, purified by affinity chromatography, were almost completely unable to catalyze the hydrolysis of the substrate. 1H-NMR spectroscopy experiments showed the absence of any major conformational changes of the three mutants with respect to the wild-type recombinant enzyme. Equilibrium dialysis experiments demonstrated that the mutated proteins lost the ability of binding inorganic phosphate, a competitive inhibitor of the enzyme. These results strongly support an involvement of arginine-23 at the phosphate binding-site of acylphosphatase, confirming the hypothesis of the existence of a phosphate binding structural motif recently proposed by other authors.
KW - (Muscle)
KW - Acylphosphatase
KW - Acylphosphatase active site
KW - NMR, H-
KW - Recombinant protein
KW - Site-directed mutagenesis
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U2 - 10.1016/0167-4838(94)90161-9
DO - 10.1016/0167-4838(94)90161-9
M3 - Article
C2 - 8086441
AN - SCOPUS:0028071719
VL - 1208
SP - 75
EP - 80
JO - Biochimica et Biophysica Acta - Protein Structure and Molecular Enzymology
JF - Biochimica et Biophysica Acta - Protein Structure and Molecular Enzymology
SN - 0167-4838
IS - 1
ER -