1GG6

CRYSTAL STRUCTURE OF GAMMA CHYMOTRYPSIN WITH N-ACETYL-PHENYLALANINE TRIFLUOROMETHYL KETONE BOUND AT THE ACTIVE SITE


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.40 Å
  • R-Value Free: 0.212 
  • R-Value Work: 0.175 

wwPDB Validation   3D Report Full Report


This is version 1.5 of the entry. See complete history


Literature

Correlation of low-barrier hydrogen bonding and oxyanion binding in transition state analogue complexes of chymotrypsin.

Neidhart, D.Wei, Y.Cassidy, C.Lin, J.Cleland, W.W.Frey, P.A.

(2001) Biochemistry 40: 2439-2447

  • DOI: https://doi.org/10.1021/bi002535a
  • Primary Citation of Related Structures:  
    1GG6, 1GGD

  • PubMed Abstract: 

    The structures of the hemiketal adducts of Ser 195 in chymotrypsin with N-acetyl-L-leucyl-L-phenylalanyl trifluoromethyl ketone (AcLF-CF3) and N-acetyl-L-phenylalanyl trifluoromethyl ketone (AcF-CF3) were determined to 1.4-1.5 A by X-ray crystallography. The structures confirm those previously reported at 1.8-2.1 A [Brady, K., Wei, A., Ringe, D., and Abeles, R. H. (1990) Biochemistry 29, 7600-7607]. The 2.6 A spacings between Ndelta1 of His 57 and Odelta1 of Asp 102 are confirmed at 1.3 A resolution, consistent with the low-barrier hydrogen bonds (LBHBs) between His 57 and Asp 102 postulated on the basis of spectroscopy and deuterium isotope effects. The X-ray crystal structure of the hemiacetal adduct between Ser 195 of chymotrypsin and N-acetyl-L-leucyl-L-phenylalanal (AcLF-CHO) has also been determined at pH 7.0. The structure is similar to the AcLF-CF3 adduct, except for the presence of two epimeric adducts in the R- and S-configurations at the hemiacetal carbons. In the (R)-hemiacetal, oxygen is hydrogen bonded to His 57, not the oxyanion site. On the basis of the downfield 1H NMR spectrum in solution, His 57 is not protonated at Nepsilon2, and there is no LBHB at pH >7.0. Because addition of AcLF-CHO to chymotrypsin neither releases nor takes up a proton from solution, it is concluded that the hemiacetal oxygen of the chymotrypsin-AcLF-CHO complex is a hydroxyl group and not attracted to the oxyanion site. The protonation states of the hemiacetal and His 57 are explained by the high basicity of the hemiacetal oxygen (pK(a) > 13.5) relative to that of His 57. The 13C NMR signal for the adduct of AcLF-13CHO with chymotrypsin is consistent with a neutral hemiacetal between pH 7 and 13. At pH <7.0, His 57 in the AcLF-CHO-hemiacetal complex of chymotrypsin undergoes protonation at Nepsilon2 of His 57, leading to a transition of the 15.1 ppm downfield signal to 17.8 ppm. The pK(a)s in the active sites of the AcLF-CF3 and AcLF-CHO adducts suggest an energy barrier of 6-7 kcal x mol(-1) against ionizations that change the electrostatic charge at the active site. However, ionizations of neutral His 57 in the AcLF-CHO-chymotrypsin adduct, or in free chymotrypsin, proceed with no apparent barrier. Protonation of His 57 is accompanied by LBHB formation, suggesting that stabilization by the LBHB overcomes the barrier to ionization. On the basis of the hydration constant for AcLF-13CHO and its inhibition constant, its K(d) is 16 microM, 8000-fold larger than the comparable value for AcLF-CF3.


  • Organizational Affiliation

    University of Wisconsin-Madison, 1710 University Avenue, Madison, Wisconsin 53705, USA.


Macromolecules

Find similar proteins by:  Sequence   |   3D Structure  

Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
GAMMA CHYMOTRYPSIN10Bos taurusMutation(s): 0 
EC: 3.4.21.1
UniProt
Find proteins for P00766 (Bos taurus)
Explore P00766 
Go to UniProtKB:  P00766
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupP00766
Sequence Annotations
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  • Reference Sequence
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Entity ID: 2
MoleculeChains Sequence LengthOrganismDetailsImage
GAMMA CHYMOTRYPSIN131Bos taurusMutation(s): 0 
EC: 3.4.21.1
UniProt
Find proteins for P00766 (Bos taurus)
Explore P00766 
Go to UniProtKB:  P00766
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupP00766
Sequence Annotations
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  • Reference Sequence
Find similar proteins by:  (by identity cutoff)  |  3D Structure
Entity ID: 3
MoleculeChains Sequence LengthOrganismDetailsImage
GAMMA CHYMOTRYPSIN97Bos taurusMutation(s): 0 
EC: 3.4.21.1
UniProt
Find proteins for P00766 (Bos taurus)
Explore P00766 
Go to UniProtKB:  P00766
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupP00766
Sequence Annotations
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  • Reference Sequence
Small Molecules
Ligands 4 Unique
IDChains Name / Formula / InChI Key2D Diagram3D Interactions
APL
Query on APL

Download Ideal Coordinates CCD File 
E [auth B]N-(1-BENZYL-3,3,3-TRIFLUORO-2,2-DIHYDROXY-PROPYL)-ACETAMIDE
C12 H14 F3 N O3
NETPVFJEHOGNPJ-JTQLQIEISA-N
APF
Query on APF

Download Ideal Coordinates CCD File 
J [auth C]1,1,1-TRIFLUORO-3-ACETAMIDO-4-PHENYL BUTAN-2-ONE(N-ACETYL-L-PHENYLALANYL TRIFLUOROMETHYL KETONE)
C12 H12 F3 N O2
JTIIYPHJIDENCW-JTQLQIEISA-N
SO4
Query on SO4

Download Ideal Coordinates CCD File 
D [auth B],
H [auth C],
I [auth C]
SULFATE ION
O4 S
QAOWNCQODCNURD-UHFFFAOYSA-L
EDO
Query on EDO

Download Ideal Coordinates CCD File 
F [auth B],
G [auth B],
K [auth C],
L [auth C]
1,2-ETHANEDIOL
C2 H6 O2
LYCAIKOWRPUZTN-UHFFFAOYSA-N
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.40 Å
  • R-Value Free: 0.212 
  • R-Value Work: 0.175 
  • Space Group: P 42 21 2
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 69.19α = 90
b = 69.19β = 90
c = 95.44γ = 90
Software Package:
Software NamePurpose
FRAMBOdata collection
XDSdata reduction
XCALIBREmodel building
X-PLORrefinement
XDSdata scaling
XCALIBREphasing

Structure Validation

View Full Validation Report



Entry History 

Deposition Data

Revision History  (Full details and data files)

  • Version 1.0: 2000-09-20
    Type: Initial release
  • Version 1.1: 2008-04-26
    Changes: Version format compliance
  • Version 1.2: 2011-07-13
    Changes: Version format compliance
  • Version 1.3: 2017-02-01
    Changes: Structure summary
  • Version 1.4: 2017-10-04
    Changes: Refinement description
  • Version 1.5: 2023-12-27
    Changes: Data collection, Database references, Derived calculations