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

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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 1H NMR spectroscopy, respectively. The N‐terminus truncated mutant lacked the first six residues (Δ6), 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 Δ6 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.