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Stability of wild-type and mutant RTEM-1 β-lactamases: Effect of the disulfide bond

Schultz, Steve C. and Dalbadie-McFarland, Gloria and Neitzel, James J. and Richards, John H. (1987) Stability of wild-type and mutant RTEM-1 β-lactamases: Effect of the disulfide bond. Proteins, 2 (4). pp. 290-297. ISSN 0887-3585. doi:10.1002/prot.340020405.

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Uniquely among class A β-lactamases, the RTEM-1 and RTEM-2 enzymes contain a single disulfide bond between Cys 77 and Cys 123. To study the possible role of this naturally occurring disulfide in stabilizing RTEM-1 β-lactamase and its mutants at residue 71, this bond was removed by introducing a Cys 77 → Ser mutation. Both the wild-type enzyme and the single mutant Cys 77 → Ser confer the same high levels of resistance to ampicillin in vivo to Echerichia coli; at 30°C the specific activity of purified Cys 77 → Ser mutant is also the same as that of the wild-type enzyme. Also, neither wild-type enzyme nor the Cys 77 → Ser mutant is inactivated by brief exposure to p-hydroxymercuri-benzoate. However, above 40°C the mutant enzyme is less stable than wild-type enzyme. After introduction of the Cys 77 → Ser mutation, none of the double mutants (containing the second mutations at residue 71) confer resistance to ampicillin in vivo at 37°C; proteins with Ala, Val, Leu, Ile, Met, Pro, His, Cys, and Ser at residue 71 confer low levels of resistance to ampicillin in vivo at 30°C. The use of electrophoretic blots stained with antibodies against β-lactamase to analyze the relative quantities of mutant proteins in whole-cell extracts of E. coli suggests that all 19 of the doubly mutant enzymes are proteolyzed much more readily than their singly mutant analogues (at Thr 71) that contain a disulfide bond. Thus, the disulfide bond of the RTEM-1 β-lactamase seems able to reduce the destabilizing effect of mutations at Thr 71. These results also emphasize the unique and essential role that Thr 71 performs in the stable folding of RTEM-1 β-lactamase and presumably the other class A β-lactamases.

Item Type:Article
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Additional Information:© 1987 Alan R. Liss, Inc. Received February 24, 1987; accepted September 22, 1987.
Subject Keywords:mutants; structural function relationships; protein folding and stability; catalysis
Issue or Number:4
Record Number:CaltechAUTHORS:20150505-092022777
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Official Citation:Schultz, S. C., Dalbadie-McFarland, G., Neitzel, J. J. and Richards, J. H. (1987), Stability of wild-type and mutant RTEM-1 β-lactamases: Effect of the disulfide bond. Proteins, 2: 290–297. doi: 10.1002/prot.340020405
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:57220
Deposited By: Tony Diaz
Deposited On:05 May 2015 16:43
Last Modified:10 Nov 2021 21:09

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