Preparation and properties of des-Tyr98 and des-Arg97-Tyr98 acylphosphatase (muscular isoenzyme).
Previous NMR reports indicated that Tyr98, the C-terminal residue of the muscular form of acylphosphatase, is likely to be part of the enzyme's active site. In addition, there is evidence that an arginine residue participates to the catalyzed reaction, possibly as phosphate binding site. Among all Arg residues present in the muscular forms of acylphosphatase, four, i.e. Arg23, Arg74, Arg77, and Arg97, appear to be conserved in all species checked thus far. We prepared the des-Tyr98 and des-Arg97-Tyr98 derivatives of the native acylphosphatase to investigate the properties of both modified enzymes. The enzyme lacking Tyr98 was found to be catalytically less effective than the native one, whereas the des-Arg97-Tyr98 acylphosphatase was completely inactive. This evidence suggests that Arg97 participates directly to the active site catalytic mechanism. Fluorescence and CD spectra revealed that the latter enzyme could have been undergone some conformational change that could account for the loss of activity; on the other hand, the one-dimensional NMR spectra of either native and des-Arg97-Tyr98 enzymes were strictly similar, thus demonstrating that the removal of the two C-terminal residues does not markedly affect the fold of the enzyme. The results reported are proof of a critical contribution of Arg97 to the acylphosphatase active site; however, we cannot exclude that the function of this residue is merely to stabilize the active site conformation and dynamics.
Stefani
M
M
Department of Molecular Biology, University of Siena, Italy.
Cappugi
G
G
Pazzagli
L
L
Camici
G
G
Manao
G
G
Taddei
N
N
Buck
M
M
Ramponi
G
G
D017766:Acid Anhydride Hydrolases; D000818:Animals; D001120:Arginine / Q000737:chemistry; D001665:Binding Sites; D002268:Carboxypeptidases / Q000737:chemistry; D002942:Circular Dichroism; D006736:Horses; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000737:chemistry* / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D009132:Muscles / Q000201:enzymology*; D010449:Peptide Mapping; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry* / Q000378:metabolism; D011489:Protein Denaturation; D014443:Tyrosine / Q000737:chemistry; D000097024:Acylphosphatase
D017766:Acid Anhydride Hydrolases; D001120:Arginine; D002268:Carboxypeptidases; D007527:Isoenzymes; D010744:Phosphoric Monoester Hydrolases; D014443:Tyrosine; D000097024:Acylphosphatase