Altering enzymatic activity: recruitment of carboxypeptidase activity into an RTEM beta-lactamase/penicillin-binding protein 5 chimera.
The D-Ala-D-Ala carboxypeptidases/transpeptidases (penicillin-binding proteins, PBPs) share considerable structural homology with class A beta-lactamases (EC 3.5.2.6), although these beta-lactamases have no observable D-Ala-D-Ala carboxypeptidase activity. With the objective of recruiting such activity into a beta-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 beta-lactamase. The segment thus inserted encompasses two residues conserved in both families: Ser-70, which forms the acyl-enzyme intermediate during beta-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 beta-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 beta-lactamases and are thought to be essential for beta-lactamase activity, the chimera has considerably reduced activity (approximately 10(-5) on penams such as penicillins and ampicillins as substrates. As a catalyst, the chimera shows an induction period of approximately 30 min, reflecting a slow conformational rearrangement from an inactive precursor to the active enzyme.
Chang
Y H
YH
Department of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena 91125.
Labgold
M R
MR
Richards
J H
JH
D000409:Alanine; D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D015153:Blotting, Western; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002678:Chimera; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D006602:Hexosyltransferases; D007700:Kinetics; D008969:Molecular Sequence Data; D002267:Muramoylpentapeptide Carboxypeptidase / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D046915:Penicillin-Binding Proteins; D010458:Peptidyl Transferases; D010957:Plasmids; D012689:Sequence Homology, Nucleic Acid; D013379:Substrate Specificity; D001618:beta-Lactamases / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D002352:Carrier Proteins; D046915:Penicillin-Binding Proteins; D010458:Peptidyl Transferases; D006602:Hexosyltransferases; D002267:Muramoylpentapeptide Carboxypeptidase; D001618:beta-Lactamases; C056342:beta-lactamase TEM-2; D000409:Alanine