Looking for residues involved in the muscle acylphosphatase catalytic mechanism and structural stabilization: role of Asn41, Thr42, and Thr46.
Asn41, Thr42, and Thr46 are invariant residues in both muscle and erythrocyte acylphosphatases isolated so far. Horse muscle acylphosphatase solution structure suggests their close spatial relationship to Arg23, the main substrate binding site. The catalytic and structural role of such residues, as well as their influence on muscle acylphosphatase stability, was investigated by preparing several gene mutants (Thr42Ala, Thr46Ala, Asn41Ala, Asn41Ser, and Asn41Gln) by oligonucleotide-directed mutagenesis. The mutated genes were cloned and expressed in Escherichia coli, and the mutant enzymes were purified by affinity chromatography and investigated as compared to the wild-type enzyme. The specific activity and substrate affinity of Thr42 and Thr46 mutants were not significantly affected. On the contrary, Asn41 mutants showed a residual negligible activity (about 0.05-0.15% as compared to wild-type enzyme), though maintaining an unchanged binding capability of both substrate and inorganic phosphate, an enzyme competitive inhibitor. According to the 1H nuclear magnetic resonance spectroscopy and circular dichroism results, all mutants elicited well-constrained native-like secondary and tertiary structures. Thermodynamic parameters, as calculated from circular dichroism data, demonstrated a significantly decreased stability of the Thr42 mutant under increasing temperatures and urea concentrations. The reported results strongly support a direct participation of Asn41 to the enzyme catalytic mechanism, indicating that Asn41 mutants may well represent a useful tool for the investigation of the enzyme physiological function by the negative dominant approach.
Taddei
N
N
Department of Biochemical Sciences, University of Florence, Italy.
Stefani
M
M
Magherini
F
F
Chiti
F
F
Modesti
A
A
Raugei
G
G
Ramponi
G
G
D017766:Acid Anhydride Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000818:Animals; D001216:Asparagine / Q000737:chemistry*; D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D002942:Circular Dichroism; D004795:Enzyme Stability; D006736:Horses; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009132:Muscles / Q000201:enzymology*; D016297:Mutagenesis, Site-Directed; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D013816:Thermodynamics; D013912:Threonine / Q000737:chemistry*; D000097024:Acylphosphatase
D017766:Acid Anhydride Hydrolases; D001216:Asparagine; D011993:Recombinant Fusion Proteins; D013912:Threonine; D000097024:Acylphosphatase