In:
Proceedings of the National Academy of Sciences, Proceedings of the National Academy of Sciences, Vol. 95, No. 11 ( 1998-05-26), p. 5971-5975
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 μM, 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 antibody–catalyst (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.
Type of Medium:
Online Resource
ISSN:
0027-8424
,
1091-6490
DOI:
10.1073/pnas.95.11.5971
Language:
English
Publisher:
Proceedings of the National Academy of Sciences
Publication Date:
1998
detail.hit.zdb_id:
209104-5
detail.hit.zdb_id:
1461794-8
SSG:
11
SSG:
12
Permalink