PDBsum entry 1scn

Hydrolase/hydrolase inhibitor

PDB id: 1scn

Contents

Protein chain

274 a.a. *

Ligands

0EF

Metals

_CA ×2

_NA

Waters ×141

* Residue conservation analysis

References listed in PDB file

Key reference

• Title: Inactivation of subtilisin carlsberg by n-((Tert-Butoxycarbonyl)alanylprolylphenylalanyl)-O-Benzoylhydroxyl- Amine: formation of a covalent enzyme-Inhibitor linkage in the form of a carbamate derivative.
• Authors: A.C.Steinmetz, H.U.Demuth, D.Ringe.
• Ref.: Biochemistry, 1994, 33, 10535-10544. [DOI no: 10.1021/bi00200a040]
• PubMed id: 8068694

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.