KINETIC AND THERMODYNAMIC CONSEQUENCES OF THE REMOVAL OF THE CYS-77-CYS-123 DISULFIDE 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 possi ble role of the disulphide bond in the stability and folding kinetics of the TEM-1 beta-lactamase, this bond was removed by introducing a Cy s-77-->Ser mutation, and the enzymically active mutant protein was stu died by reversible guanidine hydrochloride-induced denaturation. The u nfolding and refolding rates were monitored using tryptophan fluoresce nce. At low guanidine hydrochloride concentrations, the refolding of t he wild-type and mutant enzymes followed biphasic time courses. The ch aracteristics 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 pe riod and then refolded by diluting out the denaturant, indicated that, for both the wildtype and mutant enzymes, the two refolding phases co uld be ascribed to proline isomerization reactions. Equilibrium unfold ing experiments monitored by fluorescence spectroscopy and far-UV CD i ndicated a three-state mechanism (N<->H<->U). Both the folded mutant p rotein (N) and, to a lesser extent, the thermodynamically stable inter mediate, 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 in activation.