Increase of the Protease Activity of Aqualysin I, a Thermostable Serine Protease, by Replacing Asn219 near the Catalytic Residue Ser222

Abstract

Functional role of Asn219 of aqualysin I, a thermostable serine protease from Thermus aquaticus, was investigated by using site-directed mutagenesis. Replacement of Asn219 with serine increased the catalytic efficiency (k_cat/K_m) for synthetic peptide substrates about twice as much as that of the wild type, while threonine replacement caused a slight decrease in the efficiency. Such replacements resulted in a significant change of k_cat rather than K_m, indicating that the side chain in the vicinity of the catalytic residue Ser222 affects the catalytic rate constant.

Key words:

• aqualysin I
• site-directed mutagenesis
• catalytic efficiency
• protein engineering
• heat-stable protease