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 π-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 π-stacking between transition state and TrpH104.