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Published January 28, 1986 | public
Journal Article

Active-site mutants of β-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 β-lactamase (EC 3.5.2.6) with all combinations of serine and threonine residues at the active-site positions 70 and 71. (All natural β-lactamases have conserved serine-70 and threonine-71.) From the inactive double mutant Ser-70 → Thr, Thr-71 → Ser [Dalbadie-McFarland, G., Cohen, L. W., Riggs, A. D., Morin, C., Itakura, K., & Richards, J. H. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 6409-6413], an active revertant, Thr-71 → 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 → Ser) has about 15% of the catalytic activity of wild-type β-lactamase. The other possible mutant involving serine and threonine residues at positions 70 and 71 (Ser-70 → Thr) shows no catalytic activity. The primary nucleophiles of a serine or a cysteine residue [Sigal, I. S., Harwood, B. G., & 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 → Ser), we have also observed reduced thermal stability and reduced resistance to denaturation by urea.

Additional Information

© 1986 American Chemical Society. Received April 24, 1985. This work was supported by a grant from the National Institutes of Health (GM 16424). G.D.-M. and J.J.N. were supported by NRSA Grant GM 07626. We thank Steve Schultz, Arthur Riggs, and David Botstein for discussions and helpful advice and Charlotte Clark for excellent technical assistance. We also thank John Rossi and David Botstein for generously supplying bacterial strains.

Additional details

Created:
August 19, 2023
Modified:
October 23, 2023