title: Activation of adenylate kinase by denaturants is due to the increasing conformational flexibility at its active sites.
abstract: The unfolding of adenylate kinase in urea or guanidine hydrochloride solutions was measured by UV absorbance at 287 nm, circular dichroism at 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 activity of the enzyme underwent significant changes, increasing about 1.6-fold when the urea concentration was increased to 1 M. The enzyme activity then decreased with further increases of the urea concentration. We also observed that the kinetics of ANS binding to AK by fluorescence was biphasic. The fast phase completed within the dead-time of the stopped-flow apparatus used, while the slow phase ended in about 10 minutes. 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 denaturant. The change of the enzyme activity coincided with that of the rate of ANS binding during denaturation by low concentration of denaturants, suggesting that the activation of AK by denaturants may be due to the increasing conformational flexibility at its active site.
authors.affiliation: Institute of Biophysics, Academia Sinica, Beijing, 100101, China.
authors.forename: H J
authors.initials: HJ
authors.lastname: Zhang
authors.forename: X R
authors.initials: XR
authors.lastname: Sheng
authors.forename: X M
authors.initials: XM
authors.lastname: Pan
authors.forename: J M
authors.initials: JM
authors.lastname: Zhou
mesh_terms: D000263:Adenylate Kinase / Q000737:chemistry*; D000818:Animals; D004789:Enzyme Activation; D011487:Protein Conformation; D011489:Protein Denaturation; D011817:Rabbits; D013329:Structure-Activity Relationship
chemical_list: D000263:Adenylate Kinase