MECHANISM OF INHIBITION OF HUMAN-LEUKOCYTE ELASTASE BY BETA-LACTAMS .3. USE OF ELECTROSPRAY-IONIZATION MASS-SPECTROMETRY AND 2-DIMENSIONAL NMR TECHNIQUES TO IDENTIFY BETA-LACTAM-DERIVED E-I-COMPLEXES

A combination of NMR spectroscopy and electrospray ionization mass spectrometry (ESI-MS) was used to probe the identity of beta-lactam-derived complexes with serine proteases. The carbon and proton NMR chemical shifts of the human leucocyte elastase (HLE)-inhibitor complex derived from [4-C-13]-L-680,833, [S-(R*,S*)]-4-[(1-(((1-(4-methylphenyl)b tidinyl)oxy]benzeneacetic acid, were consistent with an sp(3) hybridized carbon. The ESI-MS spectrum of the L-680,833-derived HLE-I complex indicated an increase of 333 Da over the mass of the free enzyme. The data are consistent with acylation of the active site serine, loss of p-hydroxybenzeneacetic acid, and formation of a carbinolamine at the carbon deriving from C-4 of the lactam ring. The complexes produced from HLE and the diastereomers of L-680,833 display identical masses. Since the 4R-isomers produce more stable complexes [Green et al. (1995) Biochemistry 34, 14331-14343], these data suggest that these complexes differ in their stereochemistry or conformation. The structural model of the HLE-I complexes derived from the diastereomers predicts that the hydroxyl of the carbinolamine derives from a structurally observed water molecule yielding S-stereochemistry in all cases. In this model, the 4S- and 4R-diastereomers produce complexes that differ by the location of the side chain of a phenylalanine residue. The mass of HLE was increased by that of L-684,481, (R)-1-(((1-(4-methylphenyl)butyl)amino)carbonyl)-3,3-diethyl-2-azetidinone, which lacks a leaving group at C-4 in the complex derived from this compound. L-698,886, [S-(R*,S*)]-4-[(1-(((1-(4-ethoxyphenyl)b oxy]benzeneacetic acid, produces two complexes of different mass that reactivate with different rates. The mass of the less stable complex is consistent with the acyl-enzyme of 2,2-ethyl-3-oxopropanoic acid while the mass of the more stable complex is analogous to the carbinolamine observed during L-680,833 inactivation. Porcine pancreatic elastase (PPE) produces a complex with a mass consistent with replacement of the C-4 leaving group by water to produce a carbinolamine from L-684,248, [S-(R*,S*)]-4-[(1-(((1-(4-methylphenyl)butyl) amino)carbonyl)-3,3-dimethyl-2-oxo-4-azetidinyl)oxy]benzoic acid. The C-4 diastereomer, L-684,249, produces two PPE-I complexes with different masses. One of these complexes has a mass identical to the mass of the complex derived from L-684,248 while the mass of the other complex indicates the presence of the entire inhibitor molecule. The mass of the latter complex indicates that the leaving group was not liberated during the reaction. These data demonstrate that the chemical mechanism of inhibition of elastases by beta-lactams is dependent on the lactam structure. The molecular and conformational identity of the final complex controls its stability, and this is governed by the structure of the original inhibitor.