Mechanism of inhibition of human leucocyte elastase by beta-lactams. 3. Use of electrospray ionization mass spectrometry and two-dimensional NMR techniques to identify beta-lactam-derived E-I complexes.
Biochemistry
; 34(44): 14344-55, 1995 Nov 07.
Article
em En
| MEDLINE
| ID: mdl-7578038
ABSTRACT
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 [4-13C]-L-680,833, [S-(R*,S*)]-4-[(1-(((1-(4- methylphenyl)butyl)amino)carbonyl)-3,3-diethyl-2-oxo-4- azetidinyl)oxy]benzeneacetic acid, were consistent with an sp3 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-azetidino ne, which lacks a leaving group at C-4 in the complex derived from this compound. L-691,886, [S-(R*,S*)]-4-[(1-(((1-(4-ethoxyphenyl)butyl)amino)carbonyl)- 3,3-diethyl-4-oxo-2-azetidinyl)-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-dimethy l - 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.(ABSTRACT TRUNCATED AT 400 WORDS)
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Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Elastase Pancreática
/
Beta-Lactamas
Tipo de estudo:
Prognostic_studies
Limite:
Humans
Idioma:
En
Revista:
Biochemistry
Ano de publicação:
1995
Tipo de documento:
Article
País de afiliação:
Estados Unidos