Arginine-23 is involved in the catalytic site of muscle acylphosphatase.
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.
Taddei
N
N
Department of Biochemical Sciences, University of Florence, Italy.
Stefani
M
M
Vecchi
M
M
Modesti
A
A
Raugei
G
G
Bucciantini
M
M
Magherini
F
F
Ramponi
G
G
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
D017766:Acid Anhydride Hydrolases; D001120:Arginine; D000097024:Acylphosphatase