Fluoride inhibition of a purified rat liver microsomal esterase was investigated using the substrates phenyl butyrate and ethyl butyrate. The data are consistent with EHF –EH — EdES — products where S, E, EH, and EHF are substrate, active enzyme, protonated enzyme (inactive), and an inactive enzyme-fluoride complex, respectively. Fluoride binds primarily to a protonated, inactive form of the enzyme. The residue whose ionization state controls the extent of fluoride inhibition also controls catalytic activity. The residue has a pK of about 6.0 and is probably histidine. The inhibition is apparently competitive since fluoride decreases the concentration of free enzyme available for catalysis.
In the presence of fluoride, phenyl butyrate hydrolysis rates decrease slowly according to pseudo-first order kinetics, but fluoride inhibition of ethyl butyrate hydrolysis is not time dependent. Phenyl butyrate stabilizes EHF so that the apparent fluoride inhibition constant for phenyl butyrate hydrolysis is about 300 times less than for ethyl butyrate hydrolysis. In the absence of substrate, fluoride binding and release is a relatively slow process. Ethyl butyrate, however, accelerates both the rate of fluoride binding and release and does not stabilize EHF.