ABSTRACT
Antibody 16E7 catalyzes the carbon protonation of enol ether 2 to hemiacetal 3, and the carbon deprotonation of benzisoxazole 7 to phenol 8. This antibody shows an extreme case of hysteresis, requiring several hours to reach full activity. Antibody 16E7 was expressed as recombinant chimeric Fab in Escherichia coli. A model for the three-dimensional structure was produced by homology modeling and used for a docking procedure to obtain models for antibody-ligand complexes. Site-direct mutagenesis of GluL39, identified as a possible catalytic residue by the model, to either glutamine or alanine abolished catalysis, showing that both the protonation reaction of enol ether 2 and the deprotonation of benzisoxazole 7 are promoted by the same residue. The model furthermore suggested that substrate access to the catalytic site might be hindered by a flexible HCDR3 loop held in closed position by a hydrogen bond between SerH99 and GluL39, which could explain the observed hysteresis effect. In agreement with this model, mutagenesis of SerH99 to alanine, or deletion of this residue, was found to reduce hysteresis by approximately 50%.
Subject(s)
Antibodies, Catalytic/chemistry , Models, Chemical , Protons , Antibodies, Catalytic/genetics , Base Sequence , Cloning, Molecular , DNA Primers , Immunoglobulin Fab Fragments/chemistry , Immunoglobulin Fab Fragments/genetics , Kinetics , Mutagenesis, Site-DirectedABSTRACT
The cyclic ammonium cation 5 and its guanidinium analogue 4 are inhibitors of tocopherol cyclase. Monoclonal antibodies were raised against protein conjugates of the haptens 1-3 and screened for catalytic reactions with alkene 8, a short chain analogue of the natural substrate phytyl-hydroquinone 6, and its enol ether analogues 10a,b. Antibody 16E7 raised against hapten 3 was found to catalyze the hydrolysis of Z enol ether 10a to form hemiacetal 12 with an apparent rate acceleration of k(cat)/k(uncat)=1400. Antibody 16E7 also catalyzed the elimination of Kemp's benzisoxazole 59. The absence of cyclization in the reaction of enol ether 10a was attributed to the competition of water molecules for the oxocarbonium cation intermediate within the antibody binding pocket. Hapten and reaction design features contributing to this outcome are discussed. Antibody 16E7 provides the first example of a carboxyl group acting both as an acid in an intrinsically acid-catalyzed process and as a base in an intrinsically base-catalyzed process, as expected from first principles. In contrast to the many examples of general-acid-catalyzed processes known to be catalyzed by catalytic antibodies, the specific-acid-catalyzed cyclization of phytyl-hydroquinone 6 or its analogue 8 still eludes antibody catalysis.
Subject(s)
Antibodies, Catalytic/chemistry , Enzyme Inhibitors/immunology , Intramolecular Transferases/antagonists & inhibitors , Adjuvants, Immunologic/chemistry , Animals , Antibodies, Catalytic/biosynthesis , Antibodies, Catalytic/immunology , Antibodies, Monoclonal/biosynthesis , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/immunology , Catalysis , Cyclization , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Haptens/chemistry , Haptens/immunology , Hemocyanins/chemistry , Hemocyanins/immunology , Hybridomas/immunology , Intramolecular Transferases/chemistry , Intramolecular Transferases/metabolism , Kinetics , Mice , Substrate Specificity , VaccinationABSTRACT
The P450 enzyme model 1 is a high-spin system. EPR and ENDOR spectra reveal the coordination of water to the FeIII center. This is the first experimental proof that coordination of water is not the single determining factor in the stabilization of the low-spin character of the cytochrome P450 resting state.
