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

PDB id:

1scn

Name:

Hydrolase/hydrolase inhibitor

Title:

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

Structure:

Subtilisin carlsberg. Chain: e. Engineered: yes

Source:

Bacillus licheniformis. Organism_taxid: 1402

Biol. unit:

Dimer (from PQS)

Resolution:

1.90Å R-factor: 0.191

Authors:

A.C.U.Steinmetz,H.-U.Demuth,D.Ringe

Key ref:

A.C.Steinmetz et al. (1994). 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. Biochemistry, 33, 10535-10544. PubMed id: 8068694 DOI: 10.1021/bi00200a040

Date:

02-Mar-94 Release date: 31-Aug-94

Protein chain

P00780  (SUBC_BACLI) -  Subtilisin Carlsberg from Bacillus licheniformis

Seq: 379 a.a.

Struc: 274 a.a.*

* PDB and UniProt seqs differ at 3 residue positions (black crosses)

Enzyme reactions

• Enzyme class: E.C.3.4.21.62 - subtilisin.
• Reaction: Hydrolysis of proteins with broad specificity for peptide bonds, and a preference for a large uncharged residue in P1. Hydrolyzes peptide amides.

DOI no: 10.1021/bi00200a040

Biochemistry 33:10535-10544 (1994)

PubMed id: 8068694

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.

A.C.Steinmetz, H.U.Demuth, D.Ringe.

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.