UC Santa Cruz

We have specifically mutated several residues within the Class A and B. licheniformis β-lactamase sequence. These residues are believed to play an important role in either catalysis or in the structural stability of these enzymes. The main body of this thesis addresses site-directed mutagenesis studies of glutamate 166 and tyrosine 105 in the B. licheniformis β-lactamase enzyme.

Site-specific mutation of Glu-166 to Ala in β-lactamase causes a million-fold reduction in catalytic activity toward both penicillin and cephalosporin substrates, and results in the stoichiometric accumulation of a normally transient acyl-enzyme intermediate. Kinetic analysis indicated that substitution of Glu-166 by Ala leads to negligible effect on the acylation half of the reaction but effectively eliminates the deacylation reaction. Such differential effects on the rates of formation and breakdown of an enzyme-substrate intermediate have not been previously reported. Thus, unlike the situation for most transfer enzymes, e.g. the serine proteases, acylation and deacylation in β-lactamase catalysis are not "mirror" images, and must involve different mechanisms. The results suggest an explanation for the different catalytic activities between the β-lactamases and the penicillin-binding proteins involved in bacterial cell-wall synthesis.

Moreover, glutamate 166 was specifically mutated to aspartate and cysteine in order to probe the function of this residue in catalysis. In both cases, a large decrease in activity (~1.2 x 10⁵ for E166C and ~$1.7 x 10³ for E166D) was observed although the kinetics for the two mutants were very different. The pH profiles for E166D and E166C reflected the ionization characteristics of the new residue at site 166. We have interpreted this result to indicate that the deprotonation of glutamate 166 is in part responsible for the acidic limb of the W.T. activity-pH profiles. A finding which is in agreement with the hypothesized function of glutamate 166 being that of a general base. In addition, E166D was used to probe the postulated mobility of the Ω-loop upon which site 166 resides. If this region is fairly mobile then the displacement of the carboxyl functional group by 1.5 A should not significantly alter the activity. As noted above, however, the enzyme's activity is sensitive to the movement of the carboxyl functional group. A result which argues against the postulated mobility of this region. Tyrosine 105 is highly conserved throughout the Class A β-lactamase family. In order to probe the function of the hydroxyl group of this residue we mutated Tyr-105 to phenylalanine. Contrary to our expectations, the structure and activity of Y105F appeared to be unaltered when compared to the wild-type enzyme. This result may indicate that the importance of tyrosine at this site is related to the phenyl group (to a greater extent) rather than the hydroxyl group of the tyrosine residue.