title: Arginine-23 is involved in the catalytic site of muscle acylphosphatase.
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.
authors.affiliation: Department of Biochemical Sciences, University of Florence, Italy.
authors.forename: N
authors.initials: N
authors.lastname: Taddei
authors.forename: M
authors.initials: M
authors.lastname: Stefani
authors.forename: M
authors.initials: M
authors.lastname: Vecchi
authors.forename: A
authors.initials: A
authors.lastname: Modesti
authors.forename: G
authors.initials: G
authors.lastname: Raugei
authors.forename: M
authors.initials: M
authors.lastname: Bucciantini
authors.forename: F
authors.initials: F
authors.lastname: Magherini
authors.forename: G
authors.initials: G
authors.lastname: Ramponi
mesh_terms: D017766:Acid Anhydride Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001120:Arginine; D001483:Base Sequence; D001665:Binding Sites; D015153:Blotting, Western; D002384:Catalysis; D006801:Humans; D006868:Hydrolysis; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009132:Muscles / Q000201:enzymology*; D016297:Mutagenesis, Site-Directed; D013329:Structure-Activity Relationship; D000097024:Acylphosphatase
chemical_list: D017766:Acid Anhydride Hydrolases; D001120:Arginine; D000097024:Acylphosphatase