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 spe ctrometry (ESI-MS) was used to probe the identity of beta-lactam-deriv ed complexes with serine proteases. The carbon and proton NMR chemical shifts of the human leucocyte elastase (HLE)-inhibitor complex derive d from [4-C-13]-L-680,833, [S-(R,S*)]-4-[(1-(((1-(4-methylphenyl)b ti dinyl)oxy]benzeneacetic acid, were consistent with an sp(3) hybridized carbon. The ESI-MS spectrum of the L-680,833-derived HLE-I complex in dicated an increase of 333 Da over the mass of the free enzyme. The da ta are consistent with acylation of the active site serine, loss of p- hydroxybenzeneacetic acid, and formation of a carbinolamine at the car bon 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) Bio chemistry 34, 14331-14343], these data suggest that these complexes di ffer 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 wat er molecule yielding S-stereochemistry in all cases. In this model, th e 4S- and 4R-diastereomers produce complexes that differ by the locati on of the side chain of a phenylalanine residue. The mass of HLE was i ncreased by that of L-684,481, l)butyl)amino)carbonyl)-3,3-diethyl-2-a zetidinone, 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 tha t reactivate with different rates. The mass of the less stable complex is consistent with the acyl-enzyme of 2,2-ethyl-3-oxopropanoic acid w hile the mass of the more stable complex is analogous to the carbinola mine observed during L-680,833 inactivation. Porcine pancreatic elasta se (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) bonyl)-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 comp lexes has a mass identical to the mass of the complex derived from L-6 84,248 while the mass of the other complex indicates the presence of t he entire inhibitor molecule. The mass of the latter complex indicates that the leaving group was not liberated during the reaction. These d ata demonstrate that the chemical mechanism of inhibition of elastases by beta-lactams is dependent on the lactam structure. The molecular a nd conformational identity of the final complex controls its stability , and this is governed by the structure of the original inhibitor.