1Q5P

S156E/S166D variant of Bacillus lentus subtilisin


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.60 Å
  • R-Value Work: 0.168 
  • R-Value Observed: 0.169 

wwPDB Validation   3D Report Full Report


This is version 1.3 of the entry. See complete history


Literature

Do enzymes change the nature of transition states? Mapping the transition state for general acid-base catalysis of a serine protease

Bott, R.R.Chan, G.Domingo, B.Ganshaw, G.Hsia, C.Y.Knapp, M.Murray, C.J.

(2003) Biochemistry 42: 10545-10553

  • DOI: https://doi.org/10.1021/bi034773m
  • Primary Citation of Related Structures:  
    1Q5P

  • PubMed Abstract: 

    The properties of the transition state for serine protease-catalyzed hydrolysis of an amide bond were determined for a series of subtilisin variants from Bacillus lentus. There is no significant change in the structure of the enzyme upon introduction of charged mutations S156E/S166D, suggesting that changes in catalytic activity reflect global properties of the enzyme. The effect of charged mutations on the pK(a) of the active site histidine-64 N(epsilon)(2)-H was correlated with changes in the second-order rate constant k(cat)/K(m) for hydrolysis of tetrapeptide anilides at low ionic strength with a Brønsted slope alpha = 1.1. The solvent isotope effect (D)2(O)(k(cat)/K(m))(1) = 1.4 +/- 0.2. These results are consistent with a rate-limiting breakdown of the tetrahedral intermediate in the acylation step with hydrogen bond stabilization of the departing amine leaving group. There is an increase in the ratio of hydrolysis of succinyl-Ala-Ala-Pro-Phe-anilides for p-nitroaniline versus aniline leaving groups with variants with more basic active site histidines that can be described by the interaction coefficient p(xy) = delta beta(lg)/delta pK(a) (H64) = 0.15. This is attributed to increased hydrogen bonding of the active site imidazolium N-H to the more basic amine leaving group as well as electrostatic destabilization of the transition state. A qualitative characterization of the transition state is presented in terms of a reaction coordinate diagram that is defined by the structure-reactivity parameters.


  • Organizational Affiliation

    Department of Molecular Evolution and Design, Genencor International, Incorporated, 925 Page Mill Road, Palo Alto, California 94304, USA.


Macromolecules
Find similar proteins by:  (by identity cutoff)  |  3D Structure
Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
Serine protease269Lederbergia lentaMutation(s): 0 
EC: 3.4.21.62
UniProt
Find proteins for P29600 (Lederbergia lenta)
Explore P29600 
Go to UniProtKB:  P29600
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupP29600
Sequence Annotations
Expand
  • Reference Sequence
Small Molecules
Ligands 2 Unique
IDChains Name / Formula / InChI Key2D Diagram3D Interactions
SO4
Query on SO4

Download Ideal Coordinates CCD File 
B [auth A]SULFATE ION
O4 S
QAOWNCQODCNURD-UHFFFAOYSA-L
CA
Query on CA

Download Ideal Coordinates CCD File 
C [auth A],
D [auth A]
CALCIUM ION
Ca
BHPQYMZQTOCNFJ-UHFFFAOYSA-N
Modified Residues  1 Unique
IDChains TypeFormula2D DiagramParent
SEB
Query on SEB
A
L-PEPTIDE LINKINGC10 H13 N O5 SSER
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.60 Å
  • R-Value Work: 0.168 
  • R-Value Observed: 0.169 
  • Space Group: P 21 21 21
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 53.45α = 90
b = 61.5β = 90
c = 75.3γ = 90
Software Package:
Software NamePurpose
PROLSQrefinement

Structure Validation

View Full Validation Report



Entry History 

Deposition Data

Revision History  (Full details and data files)

  • Version 1.0: 2003-11-11
    Type: Initial release
  • Version 1.1: 2008-04-29
    Changes: Version format compliance
  • Version 1.2: 2011-07-13
    Changes: Version format compliance
  • Version 1.3: 2018-04-04
    Changes: Data collection