Abstract
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.
| Original language | English |
|---|---|
| Pages (from-to) | 75-80 |
| Number of pages | 6 |
| Journal | Biochimica et Biophysica Acta (BBA)/Protein Structure and Molecular |
| Volume | 1208 |
| Issue number | 1 |
| DOIs | |
| Publication status | Published - Sep 21 1994 |
Fingerprint
Keywords
- (Muscle)
- Acylphosphatase
- Acylphosphatase active site
- NMR, H-
- Recombinant protein
- Site-directed mutagenesis
ASJC Scopus subject areas
- Biochemistry
- Biophysics
- Molecular Biology
- Structural Biology
Cite this
Arginine-23 is involved in the catalytic site of muscle acylphosphatase. / Taddei, Niccolò; Stefani, Massimo; Vecchi, Manuela; Modesti, Alessandra; Raugei, Giovanni; Bucciantini, Monica; Magherini, Francesca; Ramponi, Giampietro.
In: Biochimica et Biophysica Acta (BBA)/Protein Structure and Molecular, Vol. 1208, No. 1, 21.09.1994, p. 75-80.Research output: Contribution to journal › Article
}
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
UR - http://www.scopus.com/inward/record.url?scp=0028071719&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0028071719&partnerID=8YFLogxK
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 -