1SCN

INACTIVATION OF SUBTILISIN CARLSBERG BY N-(TERT-BUTOXYCARBONYL-ALANYL-PROLYL-PHENYLALANYL)-O-BENZOL HYDROXYLAMINE: FORMATION OF COVALENT ENZYME-INHIBITOR LINKAGE IN THE FORM OF A CARBAMATE DERIVATIVE

Summary for 1SCN

• Entry DOI: 10.2210/pdb1scn/pdb
• Related PRD ID: PRD_000260
• Descriptor: SUBTILISIN CARLSBERG, N-(tert-butoxycarbonyl)-L-alanyl-N-[(1R)-1-(carboxyamino)-2-phenylethyl]-L-prolinamide, CALCIUM ION, ... (5 entities in total)
• Functional Keywords: hydrolase-hydrolase inhibitor complex, serine proteinase, hydrolase/hydrolase inhibitor
• Biological source: Bacillus licheniformis
• Cellular location: Secreted: P00780
• Total number of polymer chains: 1
• Total formula weight: 28026.05

Authors

Steinmetz, A.C.U.,Demuth, H.-U.,Ringe, D. (deposition date: 1994-03-02, release date: 1994-08-31, Last modification date: 2024-11-20)

Primary citation

Steinmetz, A.C.,Demuth, H.U.,Ringe, D.
Inactivation of subtilisin Carlsberg by N-((tert-butoxycarbonyl)alanylprolylphenylalanyl)-O-benzolhydroxyl- amine: formation of a covalent enzyme-inhibitor linkage in the form of a carbamate derivative.
Biochemistry, 33:10535-10544, 1994

PubMed Abstract: The mechanism of inactivation of serine proteases by N-peptidyl-O-aroylhydroxylamines was studied by X-ray crystallography. Cocrystals of subtilisin Carlsberg inactivated with N-((tert-butoxycarbonyl)alanylprolylphenylalanyl)-O-nitrobenzoy lhydroxylamine were grown, and diffraction data to 1.8-A resolution were obtained. The resulting electron density maps clearly reveal that the gamma-oxygen of the catalytic serine forms a carbamate derivative with the inhibitor. The peptide part of the inhibitor does not form the usual antiparallel beta-sheet in the P binding cleft but protrudes out of the active site and is stabilized by a network of water molecules. These results, combined with kinetic characterization reported previously [Demuth, H.-U., Schoenlein, C., & Barth, A. (1989b) Biochim. Biophys. Acta 996, 19-22; Schmidt, C., Schmidt, R., & Demuth, H.-U. (1990) Peptides (Giralt, E., & Andreu, D., Eds.) ESCOM Science Publishers B.V., Amsterdam] support the existence of at least one intermediate between the formation of the Michaelis complex and the final product. We suggest a mechanism for the inactivation of subtilisin Carlsberg by N-((tert-butoxycarbonyl)alanylprolylphenylalanyl)-O-benzoylhydr oxylamine whereby a negatively charged Michaelis complex undergoes a Lossen rearrangement giving rise to an isocyanate intermediate that reacts with the side chain of the active site serine.

PubMed: 8068694
DOI: 10.1021/bi00200a040

Experimental method

X-RAY DIFFRACTION (1.9 Å)