title: Evolution of aldolase antibodies in vitro: correlation of catalytic activity and reaction-based selection.
abstract: Aldolase antibodies that operate via an enamine mechanism were developed by in vitro selection. Antibody Fab phage display libraries were created where the catalytic active site residues of aldolase antibodies 38C2 and 33F12 were combined with a naive human antibody V gene repertoire. Selection from these libraries with 1,3-diketones covalently trapped the amino groups of reactive lysine residues by formation of stable enaminones. The selected aldolase antibodies retained the essential catalytic lysine residue and its function in altered and humanized primary antibody structures. The substrate specificity of the aldolase antibodies was directly related to the structure of the diketone used for selection. The k(cat) values of the antibody-catalyzed retro-aldol reactions were correlated with the K(d) values, i.e. the reactivities of the selected aldolase antibodies for the corresponding diketones. Antibodies that bound to the diketone with a lower K(d) value displayed a higher k(cat) value in the retro-aldol reaction, and a linear relationship was observed in the plots of logk(cat) versus logK(d). These results indicate that selections with diketones directed the evolution of aldolase antibodies in vitro that operate via an enamine mechanism. This strategy provides a route to tailor-made aldol catalysts with different substrate specificities.
authors.affiliation: The Skaggs Institute for Chemical Biology and Departments of Molecular Biology and Chemistry, The Scripps Research Institute, 10550 North Torrey Pines Road BCC-550, La Jolla, CA 92037, USA. ftanaka@scripps.edu
authors.forename: Fujie
authors.initials: F
authors.lastname: Tanaka
authors.forename: Roberta
authors.initials: R
authors.lastname: Fuller
authors.forename: Hyunbo
authors.initials: H
authors.lastname: Shim
authors.forename: Richard A
authors.initials: RA
authors.lastname: Lerner
authors.forename: Carlos F
authors.initials: CF
authors.lastname: Barbas
mesh_terms: D000588:Amines / Q000378:metabolism; D000595:Amino Acid Sequence; D001665:Binding Sites; D001666:Binding Sites, Antibody; D002384:Catalysis; D019020:Directed Molecular Evolution; D004797:Enzyme-Linked Immunosorbent Assay; D005634:Fructose-Bisphosphate Aldolase / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry / Q000378:metabolism*; D007659:Ketones / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D010422:Pentanones / Q000378:metabolism; D019151:Peptide Library; D016415:Sequence Alignment; D013379:Substrate Specificity
chemical_list: D000588:Amines; D007140:Immunoglobulin Fab Fragments; D007659:Ketones; D010422:Pentanones; D019151:Peptide Library; C008790:acetylacetone; C109536:antibody aldolase; D005634:Fructose-Bisphosphate Aldolase