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The Expression, Stability, and Enzymatic Activity of Specific Beta-Lactamase Mutants


Neitzel, James Joseph (1987) The Expression, Stability, and Enzymatic Activity of Specific Beta-Lactamase Mutants. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/E6R6-4J57.


Three catalytic site mutants (ser 70 to thr, thr 71 to ser, and the double mutant ser 70 to thr-thr 71 to ser) of the pBR 322 beta-lactamase were purified to homogeneity. These mutant proteins were unstable and could only be obtained from E. coli at 30°C, rather than the normal growth temperature of 37°. The use of the strong tac promoter increased the yield of the thr 71 to ser mutant protein. This promoter was coupled with a kanamycin resistance gene to create a new vector for the expression of inactive beta-lactamase mutants. A new purification protocol for the pBR 322 lactamase was devised. By replacing an early ion-exchange chromatography column with an ammonium sulfate precipitation, it was possible to isolate unstable beta-lactamases in high yield.

The ser 70 to thr mutant and the double mutant show no catalytic activity and do not form an acyl enzyme intermediate. However, they do still retain the ability to bind benzylpenicillin. The thr 71 to ser mutant is active. The Km values for benzylpenicillin and cephalothin hydrolysis are unchanged from the wild-type enzyme, while the kcat values are approximately 15% of the wild-type value. This mutant also cleaves the poor substrate cefoxitin, again with no significant change in Km, but with kcat reduced to 8% of the wild-type value. Examination of the pre-steady state burst during cefoxitin hydrolysis showed that the thr 71 to ser enzyme acylated at the same rate and to at least 80% of the extent of the wild-type enzyme. Direct measurement of the deacylation rate confirmed that a reduction in the deacylation rate is responsible for the lowered reaction rate in this mutant protein. Additionally, this protein lost catalytic activity at elevated temperature more rapidly than the wild-type enzyme.

The denaturation of the active thr 71 to ser mutant was observed in more detail. This enzyme thermally denatures at 45°, a temperature 10 to 15° lower than that required to denature the wild-type enzyme. This mutant is also more susceptible to digestion by thermolysin, trypsin, and those proteases present in vivo in the periplasmic space of E. coli. The enzyme also loses activity at a urea concentration of 2 M, whereas the wild-type enzyme is still active at urea concentrations greater than 4 M. The inactive mutants ser 70 to thr and ser 70 to thr-thr 71 to ser are even more susceptible to proteolytic attack by E. coli proteases in vivo.

A mutant pBR 322 beta-lactamase lacking the disulfide bond found in the wild-type enzyme was also purified to homogeneity. This protein showed no alterations in catalytic activity relative to the wild-type enzyme at temperatures below 40°. Above this temperature, this enzyme rapidly lost activity. This enzyme was also more susceptible to proteolytic attack at elevated temperature. However, this enzyme is more resistant to thermal and proteolytic denaturation than the thr 71 to ser mutant betalactamase.

Item Type:Thesis (Dissertation (Ph.D.))
Subject Keywords:Chemistry; Biology
Degree Grantor:California Institute of Technology
Division:Chemistry and Chemical Engineering
Major Option:Chemistry
Minor Option:Biology
Thesis Availability:Public (worldwide access)
Research Advisor(s):
  • Richards, John H.
Thesis Committee:
  • Dervan, Peter B. (chair)
  • Richards, John H.
  • Hood, Leroy E.
  • Davidson, Norman R.
Defense Date:22 December 1986
Additional Information:Thesis title listed in 1987 Commencement Program varies from actual thesis: The Expression, Stability, and Enzymatic Activity of Specific β-Lactamase Mutants.
Funding AgencyGrant Number
William Barton Jones FoundationUNSPECIFIED
Record Number:CaltechTHESIS:04162019-152444688
Persistent URL:
Default Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:11471
Deposited By: Mel Ray
Deposited On:17 Apr 2019 16:08
Last Modified:07 Nov 2019 19:55

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