Mechanism of inhibition of human leucocyte elastase by beta-lactams. 2. Stability, reactivation kinetics, and products of beta-lactam-derived E-I complexes.
The monocyclic beta-lactams reported by Knight et al. [Knight, W. B., et al. (1992) Biochemistry 31, 8160; Chabin, R., et al. (1993) Biochemistry 32, 8970] as inhibitors of human leucocyte elastase (HLE) produce stable HLE-inhibitor complexes that slowly reactivate with half-lives ranging from less than 1 to 15 h at 37 degrees C. The complexes produced between PPE and two C-3 dimethyl-substituted beta-lactams are less stable than those produced between HLE and analogous C-3 diethyl-substituted lactams. The stability of the HLE-I complexes is governed primarily by the structure of the substituted urea portion of the inhibitors and not by the identity or presence of a leaving group at C-4 of the lactam ring. In some cases substitutions on the urea portion of the inhibitors yielded complexes that displayed biphasic reactivation kinetics. This suggests the presence of at least two different complexes. The stereochemistry of the leaving group at C-4 has a small effect on the stability of the final complex (1.3-2-fold); therefore, the identity of the final complex is dependent upon the initial stereochemistry at that position. The stability of the complexes was relatively insensitive to hydroxylamine, which suggests that the acyl-enzymes are protected from nucleophilic "rescue". The rate of reactivation of the complex derived from L-680,833,[S-R*,S*)]-4-[(1-(((1-(4- methylphenyl)butyl)amino)carbonyl)-3,3-diethyl-2-oxo-4-azetidinyl)ben zeneacetic acid, was pH independent, while the L-684,481, (R)-(1-(((1-(4-methylphenyl)butyl)amino)carbonyl)-3,3-diethyl-2-azeti din one generated complex displayed a pH-dependent reactivation rate. In the latter case, the increase in reactivation rate with pH displayed a pKa of 7.2. This is consistent with the requirement for base catalysis by the active site histidine to regenerate enzymatic activity. Reactivation of the L-680,833-derived complex produced different products as a function of pH, suggesting two different pH-dependent routes of reactivation. At low pH a route that produced primarily the substituted urea is favored, while at higher pH production of two six-membered ring diastereomers competes with urea generation. Thus, the apparent pH independence of the return of activity is the result of two offsetting pathways. Other compounds such as L-670,258, (S)-4-[((((2-naphthylmethyl)amino)carbonyl)-3,3-diethyl-4-oxo-2- azetidinyl)oxy]benzoic acid, reactivate by these two routes as well as by aminolysis by the other urea nitrogen to produce an additional regioisomer. The temperature dependence of the reactivation of the complexes derived from L-684,481 and L-680,833 suggests different mechanisms.(ABSTRACT TRUNCATED AT 400 WORDS)
Green
B G
BG
Department of Enzymology, Merck Research Laboratories, Rahway, New Jersey 07065, USA.
Chabin
R
R
Mills
S
S
Underwood
D J
DJ
Shah
S K
SK
Kuo
D
D
Gale
P
P
Maycock
A L
AL
Liesch
J
J
Burgey
C S
CS
D002851:Chromatography, High Pressure Liquid; D004789:Enzyme Activation; D004793:Enzyme Reactivators / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D019272:Leukocyte Elastase; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D010196:Pancreatic Elastase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D047090:beta-Lactams / Q000378:metabolism*
D004793:Enzyme Reactivators; D047090:beta-Lactams; D010196:Pancreatic Elastase; D019272:Leukocyte Elastase