The catalytic pathway of class A ␤-lactamases involves an acyl-enzyme intermediate where the substrate is ester-linked to the Ser-70 residue. Glu-166 and Lys-73 have been proposed as candidates for the role of general base in the activation of the serine OH group. The replacement of Glu-166 by an asparagine in the TEM-1 and by a histidine in the Streptomyces albus G ␤-lactamases yielded enzymes forming stable acyl-enzymes with ␤-lactam antibiotics. Although acylation of the modified proteins by benzylpenicillin remained relatively fast, it was significantly impaired when compared to that observed with the wild-type enzyme. Moreover, the E166N substitution resulted in a spectacular modification of the substrate profile much larger than that described for other mutations of ⍀-loop residues. Molecular modeling studies indicate that the displacement of the catalytic water molecule can be related to this observation. These results confirm the crucial roles of Glu-166 and of the "catalytic" water molecule in both the acylation and the deacylation processes.DD-peptidases and most ␤-lactamases, belong to the superfamily of active-site serine penicillin-recognizing enzymes (1). The interaction between these proteins and ␤-lactams involves the formation of an acyl-enzyme (E-S * ) intermediate where the antibiotic is covalently bound to the active-site serine residue,In contrast to DD-peptidases, ␤-lactamases efficiently catalyze the deacylation step (high k 3 value) which regenerates the active enzyme and releases a biologically inactive product (P). Serine ␤-lactamases are divided into three classes A, C, and D on the basis of their primary structures. Tertiary structures of various enzymes belonging to classes A and C have been solved by x-ray crystallography underlining similarities in the folds of all these proteins (2-6). Moreover, several conserved residues were identified, some of which appear to be essential for catalysis (7). The mechanism by which serine ␤-lactamases hydrolyze penicillins and cephalosporins has received a lot of attention and, for the class A enzymes the identity of the residue involved in the activation of the active serine (Ser-70 in the ABL numbering system (8)) has been subject of controversy. Both Lys-73 (9) and Glu-166 (10) have been proposed as potential candidates for this essential role. By contrast, the function of Glu-166 in activating the hydrolytic water molecule during the deacylation step is unanimously recognized. According to Adachi et al. (11) and Strynadka et al. (9), accumulation of an acyl-enzyme during the interaction between the TEM-1 Glu-166 3 Asn mutant (E166N) and benzylpenicillin suggested that the mutation affected only the deacylation step in a severe manner. In contrast, kinetic studies of the Glu-166 3 Ala mutant of the Bacillus licheniformis ␤-lactamase (12) and of the Glu-166 3 Asp mutant of the Bacillus cereus I ␤-lactamase (13) showed that accumulation of the acyl-enzyme could result from simultaneous but different decreases of the kinetic parameters characte...

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.

The kinetics of refolding of TEM-1 beta-lactamase from solution in guanidine hydrochloride have been investigated on the manual and stopped-flow mixing time scales. The kinetics of change of far-UV circular dichroism and of intrinsic and ANS fluorescence have been compared with changes in the quenching of fluorescence by acrylamide as a probe of the accessibility of solvent to tryptophan. The binding of ANS points to hydrophobic collapse in the very early stages of folding which take place in the burst phase. This is accompanied by regain of 60-65% of native ellipticity, indicating formation of a significant proportion of secondary structure. Also in the burst phase, the tryptophan residues, which are largely exposed to solvent in the native protein, become less accessible to acrylamide, and the intrinsic fluorescence increases markedly. An early intermediate is thus formed in which tryptophan is more buried than in the native protein. Further intermediates are formed over the next 20 s. Quenching by acrylamide increases during this period, as the transient nonnative state is disrupted and the tryptophan residue(s) become(s) reexposed to solvent. The two slowest phases are determined by the isomerization of incorrect prolyl isomers, but double jump tryptophan fluorescence and acrylamide quenching experiments show little, if any, effect of proline isomerization on the earlier phases. Hydrophobic collapse thus occurs to a folding intermediate in which there is a nonnative element of structure which has to rearrange in the later steps of folding, resulting in a nonhierarchical folding pathway. The C-terminal W290 is suggested as being involved in the nonnative intermediate. beta-Lactamase provides further evidence for the occurrence of nonnative intermediates in protein folding.