Mechanism of inhibition of human leukocyte 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)

Mechanism of selective inhibition of the inducible isoform of prostaglandin G/H synthase.

“…As illustrated in Fig. 2 (15,16). By this measure the two inhibitors studied here are quite comparable in their ability to inhibit COX2: 7.76 x 10-3 and 4.26 x 10-3 sec'1-AM'1 for DuP 697 and NS-398, respectively.…”

The efficiency of C-4 substituents in activating the β-lactam scaffold towards serine proteases and hydroxide ion

“…34 For compound 5f, a linear dependence of k obs on inhibitor concentration was observed ( Figure 1B); and correction for the concentration and Michaelis constant of the substrate yielded the second-order rate constant for inhibition, (k inact /K i ), as the slope (Table 1). For compounds 5g and 5h, the individual kinetic parameters K i and k inact were obtained by determining k obs as a function of the inhibitor concentration 28 while for the remaining β-lactams (5e, 5i and 5j) the k inact /K i values were determined by calculating k obs /[I] ( Table 1). The second-order rate-constants for HLE inactivation, k inact / K i , are within the range of 10 5 to 10 6 M -1 s -1 , which reflects the stringent S 1 subsite specificity of this enzyme toward hydrophobic substituents with three or four carbon atoms (e.g.…”

“…Crystallography 26 and mass spectrometry 27 studies indicate that the reaction of monobactams containing a C-4 aryloxy substituent with HLE involves the departure of a phenol, possibly concerted with C-N bond fission and β-lactam ring-opening. 28 However, the observation that the second-order rate-constants for HLE inactivation by monobactams with no leaving group at C-4 do not differ significantly from those of monobactams containing a phenol 28 suggests that leaving group ability is not essential to inactivate the enzyme. Understanding how β-lactam substituents affects molecular recognition by the enzyme and "chemical reactivity" is most useful in designing more potent enzyme inhibitors.…”

Strategies for the inhibition of serine proteases

“…Figures 12 and 13 show the proposed mechanisms for the inhibition of HNE by a 7a-methoxysubstituted derivative and PPE by the 7a-chloro-substituted compound, respectively. Since the original discovery, a vast number of analogues of monocyclic and bicyclic b-lactams [222,224,227,228], derivatives of cephalosporins [229 -234], penems Figure 12. Proposed mechanism of inhibition of HNE by 7-methoxy-substituted cephems [222].…”