title: Mechanism-based inactivation of leukotriene A4 hydrolase during leukotriene B4 formation by human erythrocytes.
abstract: Evidence is presented in support of a mechanism-based (suicide) inactivation of leukotriene A4 hydrolyase in intact human erythrocytes by leukotriene A4 and leukotriene A4 methyl ester. Loss of enzymatic activity, accompanying leukotriene B4 formation, was proportional to the substrate concentration. Inactivation was directly related to the amount of leukotriene B4 formation: for several, different experimental protocols 50% loss of hydrolase activity corresponded with formation of 10.3 +/- 2.1 microM leukotriene B4. The time course of inactivation was pseudo-first order and obeyed saturation kinetics. Apparent inactivation (KI) and first-order rate (ki) constants for leukotriene A4 were 28 microM and 0.35 min-1, respectively. Leukotriene A4 methyl ester was also a site-directed inactivator with a similar KI = 25 microM and a ki = 0.1 min-1. For single incubations substrate instability limited the extent of inactivation to 50% of the initial enzyme activity. Following multiple, consecutive incubations with leukotriene A4 this increased and approached 80-90%; however, a residual activity of 10-20% suggested that a pool of enzyme was not susceptible to inactivation. Recovery of enzymatic activity, following inactivation, was negligible in intact erythrocytes and isolated enzyme. A single radiolabeled protein, corresponding to leukotriene A4 hydrolase, was detected by electrophoretic analysis of the incubation between [3H]leukotriene A4 and erythrocytes, or partially purified enzyme. Incorporation of [3H]leukotriene A4 methyl ester into enzyme was linearly related to its inactivation: 191 +/- 5 pmol incorporated corresponded to 10% loss of activity. Results conform to criteria for a mechanism-based inactivation, in which leukotriene A4 participates in two parallel processes, one leading to leukotriene B4 formation, the other to "suicide" inactivation of leukotriene A4 hydrolase in intact erythrocytes. The specific, rather than indiscriminate nature of this process has implications for the regulation of cellular leukotriene B4 formation. It may also afford a basis to monitor transcellular biosynthesis of leukotriene B4 in vivo.
authors.affiliation: Department of Pharmacology C-236, University of Colorado Health Sciences Center, Denver 80262.
authors.forename: L
authors.initials: L
authors.lastname: Orning
authors.forename: D A
authors.initials: DA
authors.lastname: Jones
authors.forename: F A
authors.initials: FA
authors.lastname: Fitzpatrick
mesh_terms: D004851:Epoxide Hydrolases / Q000037:antagonists & inhibitors / Q000097:blood*; D004912:Erythrocytes / Q000201:enzymology / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D017572:Leukotriene A4; D007975:Leukotriene B4 / Q000096:biosynthesis / Q000097:blood*; D015289:Leukotrienes / Q000097:blood / Q000494:pharmacology*; D013997:Time Factors
chemical_list: D017572:Leukotriene A4; D015289:Leukotrienes; D007975:Leukotriene B4; D004851:Epoxide Hydrolases; C043134:leukotriene A4 hydrolase