4QK1

Structural and Catalytic Effects of Proline Substitution and Surface Loop Deletion in the Extended Active Site of Human Carbonic Anhydrase II - K170P


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.60 Å
  • R-Value Free: 0.218 
  • R-Value Work: 0.172 
  • R-Value Observed: 0.174 

wwPDB Validation   3D Report Full Report


This is version 1.3 of the entry. See complete history


Literature

Structural and catalytic effects of proline substitution and surface loop deletion in the extended active site of human carbonic anhydrase II.

Boone, C.D.Rasi, V.Tu, C.McKenna, R.

(2015) FEBS J 282: 1445-1457

  • DOI: https://doi.org/10.1111/febs.13232
  • Primary Citation of Related Structures:  
    4QK1, 4QK2, 4QK3

  • PubMed Abstract: 

    Bioengineering of a thermophilic enzyme starting from a mesophilic scaffold has proven to be a significant challenge, as several stabilizing elements have been proposed to be the foundation of thermal stability, including disulfide bridges, surface loop reduction, ionic pair networks, proline substitutions and aromatic clusters. This study emphasizes the effect of increasing the rigidity of human carbonic anhydrase II (HCA II; EC 4.2.1.1) via incorporation of proline residues at positions 170 and 234, which are located in surface loops that are able to accommodate restrictive main-chain conformations without rearrangement of the surrounding peptide backbone. Additionally, the effect of the compactness of HCA II was examined by deletion of a surface loop (residues 230-240) that had been previously identified as a possible source of thermal stability for the hyperthermophilic carbonic anhydrase isolated from the bacterium Sulfurihydrogenibium yellowstonense YO3AOP1. Differential scanning calorimetry analysis of these HCA II variants revealed that these structural modifications had a minimum effect on the thermal stability of the enzyme, while kinetic studies showed unexpected effects on the catalytic efficiency and proton transfer rates. X-ray crystallographic analysis of these HCA II variants showed that the electrostatic potential and configuration of the highly acidic loop (residues 230-240) play an important role in its high catalytic activity. Based on these observations and previous studies, a picture is emerging of the various components within the general structural architecture of HCA II that are key to stability. These elements may provide blueprints for rational thermal stability engineering of other enzymes.


  • Organizational Affiliation

    Biochemistry & Molecular Biology, University of Florida, Gainesville, FL, USA.


Macromolecules
Find similar proteins by:  (by identity cutoff)  |  3D Structure
Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
Carbonic anhydrase 2260Homo sapiensMutation(s): 1 
Gene Names: CA2
EC: 4.2.1.1
UniProt & NIH Common Fund Data Resources
Find proteins for P00918 (Homo sapiens)
Explore P00918 
Go to UniProtKB:  P00918
PHAROS:  P00918
GTEx:  ENSG00000104267 
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupP00918
Sequence Annotations
Expand
  • Reference Sequence
Small Molecules
Ligands 1 Unique
IDChains Name / Formula / InChI Key2D Diagram3D Interactions
ZN
Query on ZN

Download Ideal Coordinates CCD File 
B [auth A]ZINC ION
Zn
PTFCDOFLOPIGGS-UHFFFAOYSA-N
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.60 Å
  • R-Value Free: 0.218 
  • R-Value Work: 0.172 
  • R-Value Observed: 0.174 
  • Space Group: P 21 21 21
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 42.012α = 90
b = 69.005β = 90
c = 73.629γ = 90
Software Package:
Software NamePurpose
DENZOdata reduction
SCALEPACKdata scaling
PHASERphasing
PHENIXrefinement
PDB_EXTRACTdata extraction
CrystalCleardata collection

Structure Validation

View Full Validation Report



Entry History 

Deposition Data

Revision History  (Full details and data files)

  • Version 1.0: 2015-03-04
    Type: Initial release
  • Version 1.1: 2015-04-29
    Changes: Database references
  • Version 1.2: 2017-11-22
    Changes: Refinement description
  • Version 1.3: 2023-09-20
    Changes: Data collection, Database references, Derived calculations, Refinement description