ACTIVATION OF ADENYLATE KINASE BY DENATURANTS IS DUE TO THE INCREASING CONFORMATIONAL FLEXIBILITY AT ITS ACTIVE-SITES

The unfolding of adenylate kinase in urea or guanidine hydrochloride s olutions was measured by UV absorbance at 287 nm, circular dichroism a t 222 nm and 8-anilino-1-naphthalenesulfonic acid (ANS) fluorescence. At concentrations less than 1.8 M of urea, the secondary and tertiary structures of AK were not noticeably perturbed. In contrast, the activ ity of the enzyme underwent significant changes, increasing about 1.6- fold when the urea concentration was increased to 1 M. The enzyme acti vity then decreased with further increases of the urea concentration. We also observed that the kinetics of ANS binding to AK by fluorescenc e was biphasic. The fast phase completed within the dead-time of the s topped-flow apparatus used, while the slow phase ended in about 10 min utes. The slow phase fluorescence rate constants increased from 0.0073 s(-1) in the absence of denaturants to 0.0100 s(-1) (about 1.4-fold) at 1 M urea and then decreased at higher urea concentrations. Similar results were obtained when guanidine hydrochloride was used as a denat urant. The change of the enzyme activity coincided with that of the ra te of ANS binding during denaturation by low concentration of denatura nts, suggesting that the activation of AK by denaturants may be due to the increasing conformational flexibility at its active site. (C) 199 7 Academic Press.