Properties of N‐terminus truncated and C‐terminus mutated muscle acylphosphatases

Abstract

Enzymatic activity and structure of N-terminus truncated and C-terminus substituted muscle acylphosphatase mutants were investigated by kinetic studies under different conditions and tH NMR spectroscopy, respectively. The N-terminus truncated mutant lacked the first six residues (A6), whereas arginine 97 and tyrosine 98 were replaced by glutamine giving two C-terminus substituted mutants (R97Q and Y98Q, respectively). All acylphosphatase forms were obtained by modifications of a synthetic gene coding for the human muscle enzyme which was expressed in E. coli. The A6 deletion mutant elicited a reduced specific activity and a native-like structure. The kinetic and structural properties of R97Q and Y98Q mutants indicate a possible role of Arg-97 in the stabilisation of the active site correct conformation, most likely via back-bone and side chain interactions with Arg-23, the residue involved in phosphate binding by the enzyme. This study also suggests a possible involvement of Tyr-98 in the stabilisation of the acylphosphatase overall structure.

C‐terminal region contributes to muscle acylphosphatase three‐dimensional structure stabilisation

“…This possibility is supported by the reduced stability of the deletion mutants reported in the present study. In another paper, it was shown that acylphosphatase Arg-97 to Gln and Tyr-98 to Gln mutants maintained over 50% and 65% of the wild-type enzyme specific activity, respectively [16]. The sharp loss of catalytic efficiency of both deletion mutants described here cannot be due to an involvement of the C-terminal residues in the catalytic mechanism.…”

“…Protein concentration in samples was made 1 2 mg/ml by dissolving the lyophilised enzymes in 50 mM d3-acetate buffer/D20, pH 3.8. The samples were immediately used for recording one-dimensional aH NMR spectra using the acquisition parameters previously reported [16]. Protein thermal unfolding was followed by recording spectra at different temperatures ranging from 30 to 70°C.…”

“…Later, a nearly complete loss of activity was shown in the muscle isozyme lacking the two C-terminal residues (Arg-97 and Tyr-98), previously removed by carboxypeptidase treatment [15]. More recently, the individual importance of Arg97 and Tyr98 for enzyme activity and stability has been studied by the same oligonucleotide-directed mutagenesis approach [16], indicating that Arg-97 and Tyr-98 do not directly participate in the catalytic mechanism. Nevertheless, these studies did not assess a possible involvement of the carboxy-terminal group in catalysis.…”

Drosophila melanogaster acylphosphatase: A common ancestor for acylphosphatase isoenzymes of vertebrate species

“…One-dimensional ~H NMR experiments were performed at 600 MHz on a AMX600 Bruker spectrometer, at 25°C as previously described [12]. The samples were prepared by five dilution/concentration cycles in 20 mM phosphate/D20 buffer, pH 7.0, containing 50 mM NaCI,…”

“…Acylphosphatase structure and site-directed mutagenesis experiments led to the proposal of a substrate-assisted catalytic mechanism [11]. In particular, the conserved residues Arg-23 and Asn-41 have been recognised as the main phosphate binding site and the catalytic residue involved in the orientation and stabilisation of catalytic water molecule, respectively [12,13]. The importance of the invariant residues belonging to the 15-21 and 42-45 loops and the role of the C-and N-terminal regions in enzyme catalysis and conformational stabilisation have also been investigated [14,15].…”