title: 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 1H NMR spectroscopy, respectively. The N-terminus truncated mutant lacked the first six residues (delta 6), 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 delta 6 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.
authors.affiliation: Department of Biochemical Sciences, University of Florence, Italy.
authors.forename: N
authors.initials: N
authors.lastname: Taddei
authors.forename: A
authors.initials: A
authors.lastname: Modesti
authors.forename: M
authors.initials: M
authors.lastname: Bucciantini
authors.forename: M
authors.initials: M
authors.lastname: Stefani
authors.forename: F
authors.initials: F
authors.lastname: Magherini
authors.forename: M
authors.initials: M
authors.lastname: Vecchi
authors.forename: G
authors.initials: G
authors.lastname: Raugei
authors.forename: G
authors.initials: G
authors.lastname: Ramponi
mesh_terms: D001483:Base Sequence; D001665:Binding Sites; D015153:Blotting, Western; D004926:Escherichia coli / Q000235:genetics; D017353:Gene Deletion; D006801:Humans; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009132:Muscles / Q000201:enzymology*; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D011994:Recombinant Proteins; D013329:Structure-Activity Relationship; D014508:Urea / Q000494:pharmacology
chemical_list: D011994:Recombinant Proteins; D014508:Urea; C018735:acetylphosphatase; D010744:Phosphoric Monoester Hydrolases