Active-site mutants of .beta.-lactamase: use of an inactive double mutant to study requirements for catalysis

Abstract

We have studied the catalytic activity and some other properties of mutants of Escherichia coli plasmid-encoded RTEM .beta.-lactamase (EC 3.5.2.6) with all combinations of serine and threonine residues at the active-site positions 70 and 71. (All natural .beta.-lactamases have conserved serine-70 and threonine-71.) From the inactive double mutant Ser-70 .fwdarw. Thr, Thr-71 .fwdarw. Ser [Dalbadie-McFarland, G., Cohen, L. W., Riggs, A. D., Morin, C., Itakura, K., and Richards, J. H. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 6409-6413], an active revertant, Thr-71 .fwdarw. Ser (i.e., residue 70 in the double mutant had changed from threonine to the serine conserved at position 70 in the wild-type enzyme), was isolated by an approach that allows identification of active revertants in the absence of a background of wild-type enzyme. This mutant (Thr-71 .fwdarw. Ser) has about 15% of the catalytic activity of wild-type .beta.-lactamase. The other possible mutant involving serine and threonine residues at positions 70 and 71 (Ser-70 .fwdarw. Thr) shows no catalytic activity. The primary nucleophiles of a serine or a cysteine residue [Sigal, I. S., Harwood, B. G., and Arentzen, R. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 7157-7160] at position 70 thus seem essential for enzymatic activity. Compared to wild-type enzyme, all three mutants show significantly reduced resistance to proteolysis; for the active revertant (Thr-71 .fwdarw. Ser), we have also observed reduced thermal stability and reduced resistance to denaturation by urea.