title: Site-Directed Chemical Mutations on Abzymes: Large Rate Accelerations in the Catalysis by Exchanging the Functionalized Small Nonprotein Components.
abstract: Taking advantage of antibody molecules to generate tailor-made binding sites, we propose a new class of protein modifications, termed as "site-directed chemical mutation." In this modification, chemically synthesized catalytic components with a variety of steric and electronic properties can be noncovalently and nongenetically incorporated into specific sites in antibody molecules to induce enzymatic activity. Two catalytic antibodies, 25E2 and 27C1, possess antigen-combining sites which bind catalytic components and act as apoproteins in catalytic reactions. By simply exchanging these components, antibodies 25E2 and 27C1 can catalyze a wide range of chemical transformations including acyl-transfer, β-elimination, aldol, and decarboxylation reactions. Although both antibodies were generated with the same hapten, phosphonate diester 1, they showed different catalytic activity. When phenylacetic acid 4 was used as the catalytic component, 25E2 efficiently catalyzed the elimination reaction of β-haloketone 2, whereas 27C1 showed no catalytic activity. In this work, we focused on the β-elimination reaction and examined the site-directed chemical mutation of 27C1 to induce activity and elucidate the catalytic mechanism. Molecular models showed that the cationic guanidyl group of ArgH52in 27C1 makes a hydrogen bond with the P═O oxygen in the hapten. This suggested that during β-elimination, ArgH52of 27C1 would form a salt bridge with the carboxylate of 4, thus destroying reactivity. Therefore, we utilized site-directed chemical mutation to change the charge properties of the catalytic components. When amine components 7-10 were used, 27C1 efficiently catalyzed the β-elimination reaction. It is noteworthy that chemical mutation with secondary amine 8 provided extremely high activity, with a rate acceleration [(kcat/Km2)/kuncat] of 1 000 000. This catalytic activity likely arises from the proximity effect, plus general-base catalysis associated the electrostatic interactions. In 27C1, the cationic guanidyl group of ArgH52is spatially close to the nitrogen of the amine components. In this microenvironment, the intrinsic pKaof the amine is perturbed and shifts to a lower pKa, which efficiently abstracts the α-proton during the reaction. This mechanism is consistent with the observed kinetic isotope effect (E2 or E1cB mechanism). Thus, site-directed chemical mutation provides a better understanding of enzyme functions and opens new avenues in biocatalyst research.
authors.affiliation: Department of Biological Science, Graduate School of Science, Osaka Prefecture University , 1-1 Gakuen-cho, Naka-ku, Sakai, Osaka 599-8531, Japan.
authors.forename: Fumihiro
authors.initials: F
authors.lastname: Ishikawa
authors.affiliation: Department of Biological Science, Graduate School of Science, Osaka Prefecture University , 1-1 Gakuen-cho, Naka-ku, Sakai, Osaka 599-8531, Japan.
authors.forename: Masato
authors.initials: M
authors.lastname: Shirahashi
authors.affiliation: Department of Biological Science, Graduate School of Science, Osaka Prefecture University , 1-1 Gakuen-cho, Naka-ku, Sakai, Osaka 599-8531, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Hayakawa
authors.affiliation: Department of Biological Science, Graduate School of Science, Osaka Prefecture University , 1-1 Gakuen-cho, Naka-ku, Sakai, Osaka 599-8531, Japan.
authors.forename: Asako
authors.initials: A
authors.lastname: Yamaguchi
authors.affiliation: Molecular Profiling Research Center for Drug Discovery (molprof), Advanced Industrial Science and Technology (AIST) , 2-4-7 Aomi, Koto-ku, Tokyo 135-0064, Japan.
authors.forename: Takatsugu
authors.initials: T
authors.lastname: Hirokawa
authors.affiliation: Department of Biological Science, Graduate School of Science, Osaka Prefecture University , 1-1 Gakuen-cho, Naka-ku, Sakai, Osaka 599-8531, Japan.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Tsumuraya
authors.affiliation: Department of Biological Science, Graduate School of Science, Osaka Prefecture University , 1-1 Gakuen-cho, Naka-ku, Sakai, Osaka 599-8531, Japan.
authors.forename: Ikuo
authors.initials: I
authors.lastname: Fujii
mesh_terms: D017151:Antibodies, Catalytic / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002384:Catalysis; D003001:Cloning, Molecular; D007700:Kinetics; D016297:Mutagenesis, Site-Directed
chemical_list: D017151:Antibodies, Catalytic