A bait and switch hapten strategy generates catalytic antibodies for phosphodiester hydrolysis.
General base catalysis supplied by the histidine-12 (H-12) residue of ribonuclease (RNase) A has long been appreciated as a major component of the catalytic power of the enzyme. In an attempt to harness the catalytic power of a general base into antibody catalysis of phosphodiester bond hydrolysis, the quaternary ammonium phosphate 1 was used as a bait and switch hapten. Based on precedence, it was rationalized that this positively charged hapten could induce a counter-charged residue in the antibody binding site at a locus suitable for it to deprotonate the 2'-hydroxyl group of the anhydroribitol phosphodiester substrate 2. After murine immunization with hapten 1, mAb production yielded a library of 35 antibodies that bound to a BSA-1 conjugate. From this panel, two were found to catalyze the cyclization-cleavage of phosphodiester 2. Kinetic studies at pH 7.49 (Hepes, 20 mM) and 25 degreesC showed that the most active antibody, MATT.F-1, obeyed classical Michaelis-Menten kinetics with a Km = 104 microM, a kcat = 0.44 min-1, and a kcat/kuncat = 1.7 x 10(3). Hapten 1 stoichiometrically inhibits the catalytic activity of the antibody. MATT.F-1 is the most proficient antibody-catalyst (1.6 x 10(7) M-1) yet generated for the function of phosphodiester hydrolysis and emphasizes the utility of the bait and switch hapten paradigm when generating antibody catalysts for processes for which general-base catalysis can be exploited.
Wentworth
P
P
Departments of Chemistry and Molecular Biology, The Scripps Research Institute and The Skaggs Institute for Chemical Biology, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.
Liu
Y
Y
Wentworth
A D
AD
Fan
P
P
Foley
M J
MJ
Janda
K D
KD
D017151:Antibodies, Catalytic / Q000737:chemistry*; D001665:Binding Sites; D006241:Haptens / Q000737:chemistry*; D006868:Hydrolysis; D007700:Kinetics; D010727:Phosphoric Diester Hydrolases / Q000737:chemistry; D013379:Substrate Specificity
D017151:Antibodies, Catalytic; D006241:Haptens; D010727:Phosphoric Diester Hydrolases