Enhancement of nuclease P1 activity in low concentration of denaturants

Abstract

Nuclease P1 is a zinc-dependent endonuclease from the mold Penicillium citrinum. Nuclease P1 exhibits an increase in activity in the presence of low concentrations of urea or GuHCl. At 0.05 M GuHCl or 1 M urea the enzyme activity enhanced maximally by 3- and 3.9-fold. The kinetic parameters indicate a decrease in K_m with an increase in catalytic constant. The K_m values for control and in presence of 0.05 M GuHCl or 1 M urea are 1.11 mg, 0.86 mg and 0.60 mg, respectively. In presence of metal ions such as Cu²⁺ and Co²⁺, urea or GuHCl treated enzyme still maintains the activity enhancement up to different extent. The far UV-CD results point to a minor conformational change and fluorescence spectra reveal increase in the relative fluorescence intensity at lower concentration. Thermal denaturation studies reveal increase in apparent T_m from 75°C for control to 80°C and 77°C in presence of 0.1 M GuHCl or 0.5 M urea, respectively. The above results show that the activation of nuclease P1 in presence of low concentrations of denaturants is mainly because of the increase in the catalytic constant suggesting that activation is due to a more open and flexible conformation of the activated enzyme with increased catalytic efficiency.