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-6809833, [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 K(i) = 0.14 and 0.06 muM, respectively. The relative potency of this class of compounds as measured by k(inact)/K(i) is primarily controlled by the K(i), 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-1 complexes. Both the pH dependence of k(inact)/K(i) for the inactivation of HLE by [S-(R*,S*)]-4-[(1-(((1-(4-methylphenyl)butyl)amino)carbonyl)-3,3-diethyl-4-oxo-2-azetidinyl)oxy]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. The latter could involve alkylation of the histidine, although this does not appear to be an absolute requirement. These data suggest that these compounds are mechanism-based inhibitors of the HLE.
