Arginine-23 is involved in the catalytic site of muscle acylphosphatase

Niccolò Taddei, Massimo Stefani, Manuela Vecchi, Alessandra Modesti, Giovanni Raugei, Monica Bucciantini, Francesca Magherini, Giampietro Ramponi

Research output: Contribution to journalArticle

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 languageEnglish
Pages (from-to)75-80
Number of pages6
JournalBiochimica et Biophysica Acta (BBA)/Protein Structure and Molecular
Volume1208
Issue number1
DOIs
Publication statusPublished - Sep 21 1994

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Arginine
Muscle
Catalytic Domain
Phosphates
Muscles
Synthetic Genes
Affinity chromatography
Mutagenesis
Dialysis
Enzyme Inhibitors
Enzymes
Site-Directed Mutagenesis
Glutamine
Affinity Chromatography
Histidine
Oligonucleotides
Nuclear magnetic resonance spectroscopy
Lysine
Hydrolysis
Magnetic Resonance Spectroscopy

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 journalArticle

Taddei, N, Stefani, M, Vecchi, M, Modesti, A, Raugei, G, Bucciantini, M, Magherini, F & Ramponi, G 1994, 'Arginine-23 is involved in the catalytic site of muscle acylphosphatase', Biochimica et Biophysica Acta (BBA)/Protein Structure and Molecular, vol. 1208, no. 1, pp. 75-80. https://doi.org/10.1016/0167-4838(94)90161-9
Taddei, Niccolò ; Stefani, Massimo ; Vecchi, Manuela ; Modesti, Alessandra ; Raugei, Giovanni ; Bucciantini, Monica ; Magherini, Francesca ; Ramponi, Giampietro. / Arginine-23 is involved in the catalytic site of muscle acylphosphatase. In: Biochimica et Biophysica Acta (BBA)/Protein Structure and Molecular. 1994 ; Vol. 1208, No. 1. pp. 75-80.
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AU - Bucciantini, Monica

AU - Magherini, Francesca

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