Expression improvement and mechanistic study of the retro-Diels-Alderase catalytic antibody 10F11 by site-directed mutagenesis.
Antibody 10F11 catalyzes the retro-Diels-Alder reaction of the bicyclic prodrug 1 releasing HNO and anthracene 4 (kcat/kuncat=2500). Earlier X-ray crystal structures of Fab 10F11 showed that tryptophan H104 at the bottom of the binding pocket interacts by pi-stacking with the aromatic ring of the substrate. Antibody 10F11 was expressed as a chimeric Fab and subjected to site-directed mutagenesis. Expression was improved by substituting a serine for a phenylalanine residue on the Fv-domain surface. Nine active-site mutants were then prepared including replacements at TrpH104, PheH101 and SerH100. Catalysis depends mainly on TrpH104 and PheH101. Catalysis is most likely caused by a combination of shape complementarity and specific electronic interactions between transition state and the aromatic residue H104. Medium and de-solvation effects have no effect on the reaction rate. Catalysis was improved to (kcat/kuncat=6300) by substituting phenylalanine for LeuL101 to indirectly enhance pi-stacking between transition state and TrpH104.
Department of Chemistry and Biochemistry, University of Berne, Freiestrasse 3, CH-3012 Berne, Switzerland.
Lei
L
Zheng
Jean-Philippe
JP
Goddard
Ulrich
U
Baumann
Jean-Louis
JL
Reymond
D017151:Antibodies, Catalytic / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D001666:Binding Sites, Antibody; D002384:Catalysis; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D006860:Hydrogen Bonding; D007128:Immunoglobulin Fragments / Q000737:chemistry; D007700:Kinetics; D008956:Models, Chemical; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010649:Phenylalanine / Q000737:chemistry; D011485:Protein Binding; D015202:Protein Engineering / Q000379:methods; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D014364:Tryptophan / Q000737:chemistry
D017151:Antibodies, Catalytic; D007128:Immunoglobulin Fragments; D011993:Recombinant Fusion Proteins; D010649:Phenylalanine; D014364:Tryptophan