Refolding of urea-denatured adenylate kinase.
The refolding of urea-denatured adenylate kinase (EC 2.7.4.3) has been followed by formation of the secondary structure, change of surface hydrophobicity and recovery of catalytic activity. During refolding of adenylate kinase with a 20-80-fold dilution of 4 M urea-denatured enzyme at 10 degrees C, the formation of the secondary structure is a fast process with a rate constant of >0.16 s-1. Transient enhancement of the 8-anilino-1-naphthalenesulphonate (ANS) fluorescence intensity is followed by a fluorescence decrease to the level equal to the value characteristic of native enzyme. The desorption of ANS binding fluorescence is relatively slow and can be fitted to a first order reaction with a rate constant of 0.004 s-1 when the ANS is present in the dilution buffer. The desorption of ANS-binding fluorescence is accelerated in the presence of nucleotide substrates. The rate constants are increased to 0.049, 0. 029, 0.028 and 0.029 s-1 in the presence of 1 mM AMP, MgATP, ATP and ADP respectively. The refolding rate constant calculated from the initial fluorescence intensity after mixing ANS with protein at different refolding intervals is 0.016 s-1, which is faster than those obtained when ANS is present throughout the refolding process, indicating that the binding of ANS with a partially folded intermediate retards its further refolding to its native structure. The reactivation rate is even faster than the rates of refolding monitored in the absence of substrates, showing that the refolding is accelerated in the presence of the substrates. A possible refolding pathway and the accelerating effect of substrates are discussed.
Zhang
H j
Hj
National Laboratory of Biomacromolecules, Institute of Biophysics, Academia Sinica, 15 Datun Road, Beijing 100101, People's Republic of China.
Sheng
X R
XR
Pan
X M
XM
Zhou
J M
JM
D000244:Adenosine Diphosphate / Q000378:metabolism; D000249:Adenosine Monophosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000263:Adenylate Kinase / Q000378:metabolism*; D000817:Anilino Naphthalenesulfonates / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013816:Thermodynamics; D014508:Urea
D000817:Anilino Naphthalenesulfonates; D005456:Fluorescent Dyes; D000249:Adenosine Monophosphate; D000244:Adenosine Diphosphate; C027132:1-anilino-8-naphthalenesulfonate; D000255:Adenosine Triphosphate; D014508:Urea; D000263:Adenylate Kinase