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


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.90 Å
  • R-Value Observed: 0.191 

wwPDB Validation   3D Report Full Report


This is version 1.4 of the entry. See complete history


Literature

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.

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

(1994) Biochemistry 33: 10535-10544

  • DOI: https://doi.org/10.1021/bi00200a040
  • Primary Citation of Related Structures:  
    1SCN

  • 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.


  • Organizational Affiliation

    Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02254.


Macromolecules
Find similar proteins by:  (by identity cutoff)  |  3D Structure
Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
SUBTILISIN CARLSBERGA [auth E]276Bacillus licheniformisMutation(s): 0 
EC: 3.4.21.62
UniProt
Find proteins for P00780 (Bacillus licheniformis)
Explore P00780 
Go to UniProtKB:  P00780
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupP00780
Sequence Annotations
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  • Reference Sequence
Small Molecules
Ligands 3 Unique
IDChains Name / Formula / InChI Key2D Diagram3D Interactions
0EF
Query on 0EF

Download Ideal Coordinates CCD File 
B [auth E]N-(tert-butoxycarbonyl)-L-alanyl-N-[(1R)-1-(carboxyamino)-2-phenylethyl]-L-prolinamide
C22 H32 N4 O6
YLCJIHHAPKJGHH-BHYGNILZSA-N
CA
Query on CA

Download Ideal Coordinates CCD File 
C [auth E],
E
CALCIUM ION
Ca
BHPQYMZQTOCNFJ-UHFFFAOYSA-N
NA
Query on NA

Download Ideal Coordinates CCD File 
D [auth E]SODIUM ION
Na
FKNQFGJONOIPTF-UHFFFAOYSA-N
Biologically Interesting Molecules (External Reference) 1 Unique
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.90 Å
  • R-Value Observed: 0.191 
  • Space Group: P 21 21 21
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 76.5α = 90
b = 55.4β = 90
c = 53.4γ = 90
Software Package:
Software NamePurpose
TNTrefinement

Structure Validation

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Entry History 

Deposition Data

Revision History  (Full details and data files)

  • Version 1.0: 1994-08-31
    Type: Initial release
  • Version 1.1: 2008-03-03
    Changes: Version format compliance
  • Version 1.2: 2011-07-13
    Changes: Atomic model, Database references, Derived calculations, Non-polymer description, Structure summary, Version format compliance
  • Version 1.3: 2012-12-12
    Changes: Other
  • Version 1.4: 2017-11-29
    Changes: Derived calculations, Other