Electrospray ionization mass spectrometry as a mechanistic tool: Mass of human leukocyte elastase and a .beta.-lactam-derived E-I complex

Abstract

We have utilized liquid chromatography electrospray ionization mass spectrometry (ESI-MS) to probe the nature of the covalent E-I complex of human leucocyte elastase (HLE) and a beta-lactam. The mass spectrum of HLE isozyme 4 displayed one major and two minor components with masses of 25,202, 25,043, and 24,522 Da, respectively. Isozyme 3 displayed three components, with masses of 25,180, 24,030, and 24,523 Da. These data suggest that the isozymes differ in the type and not the content of carbohydrate. The minor components represent decreases in carbohydrate content. Inactivation of isozyme 4 with trans-4-(ethoxycarbonyl)-3-ethyl-1-[(4-nitrophenyl)sulfonyl]-azetidin -3-one increased the mass of the three components by that of the parent compound. Similar results were obtained with the mixture of HLE isozymes. These observations demonstrate that HLE does not catalyze the beta-elimination of p-nitrophenylsulfinate as Firestone et al. [(1990) Tetrahedron 46, 2255) suggested. In addition, it suggests that a "double hit" of both the active-site serine and histidine is not required to form a stable acyl-enzyme. Noncovalent complexes between HLE and either the tight-binding secretory leucoprotease inhibitor (SLPI) or a slow tight-binding peptide difluoroketone inhibitor were not observed by ESI-MS. SLPI displayed a mass of 11,710 Da in the absence and presence of HLE. These data demonstrate the utility of ESI-MS to probe the mechanism of inhibition of enzymes by mechanism-based inhibitors.

Neutrophil Elastase as a Target in Lung Cancer

“…β-Lactams bearing leaving groups attached directly or via other functionalities to the nitrogen atom have also been proposed as HNE inhibitors [104][105][106]. In these cases highly electrophilic imine obtained by the loss of the LG was trapped by His57 to afford inactivated enzyme (Scheme (3)).…”

Electrospray ionization mass spectroscopic analysis of human erythrocyte plasma membrane phospholipids.

“…However, difficulties inherent in the structure determination and quantitation of subpicomole amounts of zwitterionic phospholipids present in biologic samples have thus far precluded facile analyses. Prior studies have demonstrated that electrospray ionization mass spectrometry (ESI-MS) allows access to a vast array of heretofore inaccessible problems in protein chemistry through dramatically enhanced volatilization of molecular ions (17)(18)(19)(20). However, to date, the utility of ESI-MS for the structure identification and quantitative analysis of phospholipids present in biologic membranes has not been realized.…”

Synthetic Inhibitors of Elastase

“…A conformational rearrangement of the initially formed adduct may protect the acyl-enzyme from hydrolysis. 258 The azetidinone inhibitors of HNE are proposed to bind within the active-site in a manner similar to that of the cephalosporins, with the substituent on the C-2 carbon occupying the S, subsite and the nitrogen substituent extending into the S,'-S,' subsites. 259 In contrast to cephalosporins where C-7p substituents were detrimental to binding in the s, subsite, disubstitution is tolerated at C-2 of the azetidinones, possibly due to the increased flexibility of the monocyclic system within the active-site.…”