Schultz, Steve C. and Richards, John H. (1986) Site-saturation studies of β-lactamase: Production and characterization of mutant β-lactamases with all possible amino acid substitutions at residue 71. Proceedings of the National Academy of Sciences of the United States of America, 83 (6). pp. 1588-1592. ISSN 0027-8424. http://resolver.caltech.edu/CaltechAUTHORS:SCHUpnas86
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A mutagenic technique that "saturates" a particular site in a protein with all possible amino acid substitutions was used to study the role of residue 71 in β-lactamase (EC 184.108.40.206). Threonine is conserved at residue 71 in all class A β-lactamases and is adjacent to the active site Ser-70. All 19 mutants of the enzyme were characterized by the penam and cephem antibiotic resistance they provided to Escherichia coli LS1 cells. Surprisingly, cells producing any of 14 of the mutant β-lactamases displayed appreciable resistance to ampicillin; only cells with mutants having Tyr, Trp, Asp, Lys, or Arg at residue 71 had no observable resistance to ampicillin. However, the mutants are less stable to cellular proteases than wild-type enzyme is. These results suggest that Thr-71 is not essential for binding or catalysis but is important for stability of the β-lactamase protein. An apparent change in specificity indicates that residue 71 influences the region of the protein that accommodates the side chain attached to the β-lactam ring of the substrate.
|Additional Information:||© 1986 by the National Academy of Sciences. Communicated by Norman Davidson, October 29, 1985. We thank Suzanne Horvath and Marilyn Tomich for synthesis of the oligonucleotides. We thank Gloria Dalbadie-McFarland for her help on the oligonucleotide-directed mutagenesis, Mingjim Wu for his laboratory assistance, and James Neitzel for providing the anti-β-lactamase antibody. We also thank Gloria Dalbadie-McFarland, James Neitzel, Steven Carroll, Yie-Hwa Chang, and William Healey for many helpful discussions and suggestions. This work was supported by National Institutes of Health Grant GM 16424. This is contribution no. 7282 from the Division of Chemistry and Chemical Engineering, California Institute of Technology. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.|
|Subject Keywords:||mutagenesis; enzymatic catalysis; protein structure-function relationships; protein stability|
|Usage Policy:||No commercial reproduction, distribution, display or performance rights in this work are provided.|
|Deposited By:||Tony Diaz|
|Deposited On:||19 May 2008|
|Last Modified:||26 Dec 2012 10:02|
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