Open AccessHalf‐of‐the‐sites reactivity of outer‐membrane phospholipase A against an active‐site‐directed inhibitor
Author(s) -
UbarretxenaBelandia Iban,
Cox Ruud C.,
Dijkman Ruud,
Egmond Maarten R.,
M. Verheij Hubertus,
Dekker Niek
Publication year - 1999
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1046/j.1432-1327.1999.00221.x
Subject(s) - active site , chemistry , enzyme , stereochemistry , reactivity (psychology) , enzyme inhibitor , phospholipase a2 , phospholipase a , phospholipase , membrane , biochemistry , medicine , alternative medicine , pathology
The reaction of a novel active‐site‐directed phospholipase A 1 inhibitor with the outer‐membrane phospholipase A (OMPLA) was investigated. The inhibitor 1‐ p ‐nitrophenyl‐octylphosphonate‐2‐tridecylcarbamoyl‐3‐ethanesulfonyl‐amino‐3‐deoxy‐ sn ‐glycerol irreversibly inactivated OMPLA. The inhibition reaction did not require the cofactor calcium or an unprotonated active‐site His142. The inhibition of the enzyme solubilized in hexadecylphosphocholine micelles was characterized by a rapid ( t 1/2 = 20 min) and complete loss of enzymatic activity, concurrent with the covalent modification of 50% of the active‐site serines, as judged from the amount of p ‐nitrophenolate (PNP) released. Modification of the remaining 50% occurred at a much lower rate, indicative of half‐of‐the‐sites reactivity against the inhibitor of this dimeric enzyme. Inhibition of monomeric OMPLA solubilized in hexadecyl‐ N,N‐ dimethyl‐1‐ammonio‐3‐propanesulfonate resulted in an equimolar monophasic release of PNP, concurrent with the loss of enzymatic activity ( t 1/2 = 14 min). The half‐of‐the‐sites reactivity is discussed in view of the dimeric nature of this enzyme.