MECHANISM OF BIEXPONENTIAL INACTIVATION OF ORGANOPHOSPHATE HYDROLASE BY 1,10-PHENANTROLINE - A KINETIC AND 2ND-DERIVATIVE UV SPECTRAL STUDY

Gentle thermoinactivation of cobalt(II)-dependent organophosphate hydr olase isolated from E. coli DH5 alpha at 25 degrees C and pH 9.0 bring s about (i) biexponential loss of the enzymatic activity, which is bel ieved to be due to consecutive removal of two metal ions from the enzy me active site by 1,10-phenantroline (phen) and (ii) the rate law chan geover. The rare of both steps becomes independent of [phen] and the c orresponding rate constants k(1) and k(2) equal (4.6 +/- 0.7) x 10(-3) and (1.5 +/- 0.2) x 10(-4) s(-1), respectively. The changeover was fo llowed by measuring the second derivative UV spectra of the same prepa ration of the enzyme and changes in lambda(max) are indicative of its marginal unfolding. Likely, the unfolding distorts the favorable tetra hedral coordinative environment around metals thus facilitating their dissociation.