Activation of chicken liver dihydrofolate reductase in concentrated urea solutions.
The activation and inactivation of dihydrofolate reductase from chicken liver during denaturation in a wide concentration range of urea are compared with changes in intrinsic fluorescence. At 2 M urea the enzyme is activated 3.6-fold and is stable up to 12 h in the activated form. At 4 M urea, the enzyme activity increases about 5-fold initially but the activated enzyme loses activity rapidly to a level well below that of the native enzyme. The activated enzyme is stabilized in presence of either DHF or NADPH. The Kd and Km of the enzyme for the substrates at various urea concentrations were determined and compared. In the presence of 3 M urea, the values of Kd for DHF and NADPH increase 4-fold and 10-fold, respectively, whereas the corresponding Km values increase 25-fold and 3-fold. A large increase in Vmax is mainly responsible for the activation. The inactivation and unfolding in urea are both biphasic processes. For the fast phase, the rate constant of inactivation is 10-fold greater than that of unfolding in 4 M urea. The effect of (NH4)2SO4 on the activation and unfolding of the enzyme was also studied. The results suggest that the active site of the enzyme is more easily perturbed by denaturants; and the activated enzyme appears to have a more open and flexible conformation at the active site, which is favorable for the full expression of the catalytic power of the enzyme. A scheme for the sequential activation and inactivation of DHFR accompanying its unfolding by increasing concentrations of urea is proposed.
Fan
Y X
YX
National Laboratory of Biomacromolecules, Institute of Biophysics, Academia Sinica, Beijing, China.
Ju
M
M
Zhou
J M
JM
Tsou
C L
CL
D000645:Ammonium Sulfate; D000818:Animals; D001665:Binding Sites; D002645:Chickens; D004789:Enzyme Activation / Q000187:drug effects; D005453:Fluorescence; D008099:Liver / Q000201:enzymology*; D011487:Protein Conformation; D012996:Solutions; D013762:Tetrahydrofolate Dehydrogenase / Q000378:metabolism*; D014508:Urea / Q000494:pharmacology*
D012996:Solutions; D014508:Urea; D013762:Tetrahydrofolate Dehydrogenase; D000645:Ammonium Sulfate