Kinetic and thermodynamic consequences of the removal of the Cys-77-Cys-123 disulphide bond for the folding of TEM-1 beta-lactamase.
Class A beta-lactamases of the TEM family contain a single disulphide bond which connects cysteine residues 77 and 123. To clarify the possible role of the disulphide bond in the stability and folding kinetics of the TEM-1 beta-lactamase, this bond was removed by introducing a Cys-77-->Ser mutation, and the enzymically active mutant protein was studied by reversible guanidine hydrochloride-induced denaturation. The unfolding and refolding rates were monitored using tryptophan fluorescence. At low guanidine hydrochloride concentrations, the refolding of the wild-type and mutant enzymes followed biphasic time courses. The characteristics of the two phases were not significantly affected by the mutation. Double-jump experiments, in which the protein was unfolded in a high concentration of guanidine hydrochloride for a short time period and then refolded by diluting out the denaturant, indicated that, for both the wild-type and mutant enzymes, the two refolding phases could be ascribed to proline isomerization reactions. Equilibrium unfolding experiments monitored by fluorescence spectroscopy and far-UV CD indicated a three-state mechanism (N<-->H<--U). Both the folded mutant protein (N) and, to a lesser extent the thermodynamically stable intermediate, H. were destabilized relative to the fully unfolded state, U. Removal of the disulphide bond resulted in a decrease of 14.2 kJ/mol (3.4 kcal/mol) in the global free energy of stabilization. Similarly, the mutation also induced a drastic increase in the rate of thermal inactivation.
Vanhove
M
M
Laboratoire d'Enzymologie, Institut de Chimie B6, Université de Liège, Belgium.
Guillaume
G
G
Ledent
P
P
Richards
J H
JH
Pain
R H
RH
Frère
J M
JM
D004220:Disulfides; D004355:Drug Stability; D004789:Enzyme Activation / Q000235:genetics; D006358:Hot Temperature; D007700:Kinetics; D009154:Mutation; D017510:Protein Folding; D017384:Sequence Deletion; D013816:Thermodynamics; D001618:beta-Lactamases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
D004220:Disulfides; D001618:beta-Lactamases