title: Half-of-the-sites reactivity of outer-membrane phospholipase A against an active-site-directed inhibitor.
abstract: The reaction of a novel active-site-directed phospholipase A1 inhibitor with the outer-membrane phospholipase A (OMPLA) was investigated. The inhibitor 1-p-nitrophenyl-octylphosphonate-2-tridecylcarbamoyl-3-et hanesulfonyl -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 (t1/2 = 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 (t1/2 = 14 min). The half-of-the-sites reactivity is discussed in view of the dimeric nature of this enzyme.
authors: Department of Enzymology and Protein Engineering, Utrecht University, The Netherlands.
authors: I
authors: I
authors: Ubarretxena-Belandia
authors: R C
authors: RC
authors: Cox
authors: R
authors: R
authors: Dijkman
authors: M R
authors: MR
authors: Egmond
authors: H M
authors: HM
authors: Verheij
authors: N
authors: N
authors: Dekker
mesh_terms: D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000138:chemical synthesis / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D010741:Phospholipases A / Q000037:antagonists & inhibitors* / Q000378:metabolism; D054466:Phospholipases A1; D010743:Phospholipids / Q000138:chemical synthesis / Q000494:pharmacology*; D013210:Staphylococcus / Q000201:enzymology; D013379:Substrate Specificity
chemical_list: C118645:1-nitrophenyl-octylphosphonate-2-tridecylcarbamoyl-3-ethanesulfonylamino-3-deoxyglycerol; D004791:Enzyme Inhibitors; D010743:Phospholipids; D010741:Phospholipases A; D054466:Phospholipases A1; D002118:Calcium