A bait and switch hapten strategy generates catalytic antibodies for phosphodiester hydrolysis

Abstract

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°C showed that the most active antibody, MATT.F-1, obeyed classical Michaelis-Menten kinetics with a K m ‫؍‬ 104 M, a k cat ‫؍‬ 0.44 min ؊1 , and a k cat ͞k uncat ‫؍‬ 1.7 ؋ 10 3 . Hapten 1 stoichiometrically inhibits the catalytic activity of the antibody. MATT.F-1 is the most proficient antibodycatalyst (1.6 ؋ 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.The hydrolysis of phosphodiester bonds, such as those found in DNA and RNA, is a reaction of fundamental importance in living systems. Consequently, there are intense efforts centered around the development of novel phosphodiesterases for use in biochemistry and medicine (1-5). Our approach to this area has been to exploit the diversity of the murine immune system (6, 7) to generate antibodies possessing phosphodiesterase activity. Preliminary success in this area was based on our work with oxotechnetium(v) and oxorhenium(V) complexes that inhibit ribonuclease (RNase) U 2 (EC 3.1.27.4) activity (8-10). The oxorhenium(v) hapten 3, a putative transition state analog for the cyclization-cleavage of oligonucleotides, generated a catalytic antibody, 2G12, that catalyzes the hydrolysis of uridine 3Ј-(p-nitrophenyl) phosphate (UpOC 6 H 4 -p-NO 2 ) 4 (11) (Fig. 1).The most studied of the natural ribonucleases is bovine pancreatic RNase A (EC 3.1.27.5) (12-14). X-ray diffraction (15), chemical modification (16), and pH-rate studies (17) are all consistent with an enzymatic reaction mechanism in which the rate-limiting transition state for RNA cleavage is similar to that shown in Fig. 2.Although this mechanism has stimulated much speculation in the fields of bioorganic chemistry and enzymology (18)(19)(20)(21)(22), it does seem clear that the imidazole group of histidine 12 (H-12) acts as a general base by deprotonating the 2Ј oxygen and that the imidazolium group of histidine 119 (H-119) acts as a general acid by either protonating the 5ЈЈ oxygen of the leaving group (classical mechanism) or a non...

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