title: Mechanism of inhibition of human leucocyte elastase by monocyclic beta-lactams.
abstract: The kinetic and catalytic mechanisms of time-dependent inhibition of human polymorphonuclear leukocyte elastase (HLE) by the monocyclic beta-lactams described by Knight et al. [Knight, W.B., et al. (1992) Biochemistry 31, 8160] are investigated in this work. The dependence of the pseudo-first-order rate constant (k(obs)) on inhibitor concentration was saturable. The individual kinetic constants for the inhibition by L-680,833, [S-(R*,S*)]-4-[(1-(((1-(4- methylphenyl)butyl)amino)carbonyl)-3,3-diethyl-4-oxo-2- azetidinyl)oxy]benzeneacetic acid, and L-683,845, [S-(R*,S*)]-4-[(1-(((1-(5-benzofuranyl)butyl)amino)carbonyl)- 3,3-diethyl-4-oxo-2-azetidinyl)oxy]benzeneacetic acid, at pH 7.5 were k(inact) = 0.08 and 0.06 s-1 and Ki = 0.14 and 0.06 microM, respectively. The relative potency of this class of compounds as measured by k(inact)/Ki is primarily controlled by the Ki, term which ranged from 6 nM to 8 mM, while K(inact) was relatively insensitive to structural changes and varied by only an order of magnitude. Inactivation by the beta-lactams was efficient, requiring only 1.3 and 1.7 equiv of L-680,833 and L-683,845 to inactivate HLE. These values are indicative of some partitioning between turnover of inhibitor and inactivation. The partition ratio ranged as high as 3.5:1 depending upon the structure of the inhibitors, but this ratio was essentially independent of the availability and identity of a leaving group at C-4 of the lactam ring. Inactivation and partitioning liberate the leaving group when present at C-4. p-Hydroxy-m-nitrophenylacetic acid is liberated from this position at a rate similar to that for enzyme inactivation, suggesting kinetic competence of this process. Other products observed during the interaction of L-680,833 with HLE include a substituted urea, a species previously observed during the base-catalyzed decomposition of this class of compounds, and small amounts of products observed during reactivation of beta-lactam-derived HLE-I complexes. Both the pH dependence of k(inact)/Ki for the inactivation of HLE by [S-(R*,S*)]-4-[(1-(((1-(4-methylphenyl)butyl)amino)carbonyl)-3,3-diethyl - 4-oxo-2-azetidinyl)oxyl]benzoic acid and V/K for HLE-catalyzed substrate hydrolysis indicate that a single ionizable group with a pK of approximately 7 must be deprotonated for both processes. This group is likely the active site histidine. The data are consistent with initial formation of a Michaelis complex, acylation of the catalytic serine, and loss of the leaving group at C-4 of the original beta-lactam ring followed by partitioning between regeneration of active enzyme and production of a stable enzyme-inhibitor complex.(ABSTRACT TRUNCATED AT 400 WORDS)
authors.affiliation: Department of Enzymology, Merck Research Laboratories, Rahway, New Jersey 07065.
authors.forename: R
authors.initials: R
authors.lastname: Chabin
authors.forename: B G
authors.initials: BG
authors.lastname: Green
authors.forename: P
authors.initials: P
authors.lastname: Gale
authors.forename: A L
authors.initials: AL
authors.lastname: Maycock
authors.forename: H
authors.initials: H
authors.lastname: Weston
authors.forename: C P
authors.initials: CP
authors.lastname: Dorn
authors.forename: P E
authors.initials: PE
authors.lastname: Finke
authors.forename: W K
authors.initials: WK
authors.lastname: Hagmann
authors.forename: J J
authors.initials: JJ
authors.lastname: Hale
authors.forename: M
authors.initials: M
authors.lastname: MacCoss
mesh_terms: D002384:Catalysis; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D019272:Leukocyte Elastase; D010196:Pancreatic Elastase / Q000037:antagonists & inhibitors*; D047090:beta-Lactams / Q000494:pharmacology*
chemical_list: D047090:beta-Lactams; D010196:Pancreatic Elastase; D019272:Leukocyte Elastase