Reaction of a microsomal esterase from hog-liver with diethyl rho-nitrophenyl phosphate.

Abstract

Abstract 1. 1. A study has been made on the reaction of a highly purified carboxylesterase (carboxylic-ester hydrolase, EC 3.1.1.1) from hog-liver microsomes with diethyl p- nitrophenyl phosphate. 2. 2. Both p- and o- nitrophenyl acetates are hydrolysed by hog-live esterase, the turnover numbers for these substrates being 40 000–50 000. These figures are more than 4 orders of magnitude greater than the corresponding turnover numbers for chymotrypsin. 3. 3. Hog-liver estarase reacts very rapidly with diethyl p- nitrophenyl phosphate as indicated by the burst-like liberation of a stoichiometric amount of p- nitrophenol . 4. 4. The diethyl p- nitrophenyl phosphate-inhibited liver estarase is slowly reactivated. The recovery of the free enzyme follows the kinetics of a first-order reaction. 5. 5. One mole of p- nitrophenol is liberated from an excess of diethyl p- nitrophenyl phosphate by 86 300 ± 10 800 g of esterase protein. 6. 6. In titration experiments with stoichiometric amount of diethyl p- nitrophenyl phosphate the activities towards acetanilide, procaine, phenacetin and l -leucyl-β-naphthylamide are just completely inhibited when 1 mole of enzyme has reacted with 2 moles of the inhibitor. With an excess of enzyme each mole of diethyl p- nitrophenyl phosphate added yields 1 mole of p- nitrophenol . 7. 7. It is concluded that 1 molecule of hog-liver esterase contains 2 active centres and that the hydrolysis of the amide and ester substrates mentioned is catalysed by the same site.