C-terminal region contributes to muscle acylphosphatase three-dimensional structure stabilisation.
Ser-Ala and Ser-Ala-Ser-Ala C-terminus elongated (delta+2 and delta+4, respectively) and two C-terminus deleted (delta-2 and delta-3) muscle acylphosphatase mutants were investigated to assess the catalytic and structural roles of the C-terminal region. The kinetic analysis of these mutants shows that the removal of two or three C-terminal residues reduces the catalytic activity to 7% and 4% of the value measured for the wild-type enzyme, respectively; instead, the elongation of the C-terminus does not significantly change the enzyme behaviour. 1H Nuclear magnetic resonance spectroscopy indicates that all mutants display a native-like fold though they appear less stable, particularly delta-2 and delta-3 mutants, as compared to the wild-type enzyme. Such destabilisation of the C-terminal modified mutants is further confirmed by urea inactivation experiments. The results here presented account for an involvement of the C-terminal region in the stabilisation of the three-dimensional structure of acylphosphatase, particularly at the active-site level. Moreover, a participation of the C-terminal carboxyl group to the catalytic mechanism can be excluded.
Taddei
N
N
Department of Biochemical Sciences, University of Florence, Italy.
Magherini
F
F
Chiti
F
F
Bucciantini
M
M
Raugei
G
G
Stefani
M
M
Ramponi
G
G
D017766:Acid Anhydride Hydrolases / Q000737:chemistry* / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D005813:Genes, Synthetic; D006736:Horses; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000737:chemistry* / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D013329:Structure-Activity Relationship; D000097024:Acylphosphatase
D017766:Acid Anhydride Hydrolases; D009124:Muscle Proteins; D011993:Recombinant Fusion Proteins; D000097024:Acylphosphatase