Taddei N, Stefani M, Vecchi M, Modesti A, Raugei G, Bucciantini M, Magherini F, Ramponi G
Department of Biochemical Sciences, University of Florence, Italy.
Biochim Biophys Acta. 1994 Sep 21;1208(1):75-80. doi: 10.1016/0167-4838(94)90161-9.
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.
