The modification of cholinesterase activity by 5, 5′-dithiobis-(2-nitrobenzoic acid) included in the coupled spectrophotometric assay. Evidence for a non-catalytic substrate-binding site

Abstract

1. Compared with the acetylcholinesterase assay carried out in the absence of a dithiol, the presence of 5,5′-dithiobis-(2-nitrobenzoic acid) caused marked activation, 6,6′-dithiodinicotinic acid and 2,2′-dithiobis-(5-nitropyridine) less so and 2,2′-dithiodipyridine (aldrithiol-2) had no effect at all. Measurements are further complicated in that the 5-thio-2-nitrobenzoate ion also appears to interact with the enzyme, resulting in slightly lowered absorbance values. 2. Acetylthiocholine competes for the 5,5′-dithiobis-(2-nitrobenzoic acid)-binding site so that activation is essentially eliminated by saturating concentrations of substrate. The presence of the dithiol decreases the K_m value of acetylthiocholine. 3. Similar results were obtained with pseudocholinesterase. However, with butyrylthiocholine clear activation was still observed under V_max. conditions in addition to K_m being lowered. 4. All the data yielded Hill coefficients of 1 and analysis of the results leads to the conclusion that activation results from the dithiol being bound to a site on the subunit that is actively catalysing ester hydrolysis. 5. The use of aldrithiol-2 is recommended for kinetic work where absolute quantitative measurements are required.