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Altering Enzymatic Activity: Recruitment of Carboxypeptidase Activity into an RTEM β-Lactamase/Penicillin-Binding Protein 5 Chimera

Chang, Yie-Hwa and Labgold, Marc R. and Richards, John H. (1990) Altering Enzymatic Activity: Recruitment of Carboxypeptidase Activity into an RTEM β-Lactamase/Penicillin-Binding Protein 5 Chimera. Proceedings of the National Academy of Sciences of the United States of America, 87 (7). pp. 2823-2827. ISSN 0027-8424. PMCID PMC53783. doi:10.1073/pnas.87.7.2823.

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The D-Ala-D-Ala carboxypeptidases/transpeptidases (penicillin-binding proteins, PBPs) share considerable structural homology with class A β-lactamases (EC, although these β-lactamases have no observable D-Ala-D-Ala carboxypeptidase activity. With the objective of recruiting such activity into a β-lactamase background, we have prepared a chimeric protein by inserting a 28-amino acid segment of PBP-5 of Escherichia coli in place of the corresponding region of the RTEM-1 β-lactamase. The segment thus inserted encompasses two residues conserved in both families: Ser-70, which forms the acyl-enzyme intermediate during β-lactam hydrolysis, and Lys-73, whose presence has been shown to be necessary for catalysis. This chimera involves changes of 18 residues and gives a protein that differs at 7% of the residues from the parent. Whereas RTEM β-lactamase has no D-Ala-D-Ala carboxypeptidase activity, that of the chimera is significant and is, in fact, about 1% the activity of PBP-5 on diacetyl-L-Lys-D-Ala-D-Ala; in terms of free energy of activation, the chimera stabilizes the transition state for the reaction to within about 2.7 kcal/mol of the stabilization achieved by PBP-5. Furthermore, the chimera catalyzes hydrolysis exclusively at the carboxyl-terminal amide bond which is the site of cleavage by D-Ala-D-Ala carboxypeptidase. Though containing all those residues that are conserved throughout class A β-Lactamases and are thought to be essential for β-lactamase activity, the chimera has considerably reduced activity ({approx} 10^-5) on penams such as penicillins and ampicillins as substrates. As a catalyst, the chimera shows an induction period of {approx} 30 min, reflecting a slow conformational rearrangement from an inactive precursor to the active enzyme.

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Additional Information:© 1990 by National Academy of Sciences. Communicated by Norman Davidson, January 2, 1990 (received for review October 1, 1989) This research was supported by Grant GM16424 from the National Institutes of Health, by the Caltech Consortium in Chemistry, and by the Office of Naval Research. This is contribution no. 7935 of 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.
Funding AgencyGrant Number
Caltech Consortium in Chemistry and Chemical EngineeringUNSPECIFIED
Office of Naval Research (ONR)UNSPECIFIED
Subject Keywords:mutagenesis; protein structure-function relationships; enzymatic catalysis
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Caltech Division of Chemistry and Chemical Engineering7935
Issue or Number:7
PubMed Central ID:PMC53783
Record Number:CaltechAUTHORS:CHApnas90b
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Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:9909
Deposited By: Archive Administrator
Deposited On:26 Mar 2008
Last Modified:08 Nov 2021 21:03

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