2WUV

Crystallographic analysis of counter-ion effects on subtilisin enzymatic action in acetonitrile

Summary for 2WUV

• Entry DOI: 10.2210/pdb2wuv/pdb
• Related: 1AV7 1AVT 1C3L 1OYV 1R0R 1SBC 1SCN 1SEL 1VSB 1YU6 2SEC 2WUW 3VSB
• Descriptor: SUBTILISIN CARLSBERG, CHLORIDE ION, CESIUM ION, ... (7 entities in total)
• Functional Keywords: serine protease, metal-binding, hydrolase
• Biological source: BACILLUS LICHENIFORMIS
• Cellular location: Secreted: P00780
• Total number of polymer chains: 1
• Total formula weight: 29320.11

Authors

Cianci, M.,Tomaszewki, B.,Helliwell, J.R.,Halling, P.J. (deposition date: 2009-10-09, release date: 2010-12-08, Last modification date: 2023-12-20)

Primary citation

Cianci, M.,Tomaszewski, B.,Helliwell, J.R.,Halling, P.J.
Crystallographic Analysis of Counterion Effects on Subtilisin Enzymatic Action in Acetonitrile.
J.Am.Chem.Soc., 132:2293-, 2010

PubMed Abstract: When enzymes are in low dielectric nonaqueous media, it would be expected that their charged groups would be more closely associated with counterions. There is evidence that these counterions may then affect enzymatic activity. Published crystal structures of proteins in organic solvents do not show increased numbers of associated counterions, and this might reflect the difficulty of distinguishing cations like Na(+) from water molecules. In this paper, the placement of several Cs(+) and Cl(-) ions in crystals of the serine protease subtilisin Carlsberg is presented. Ions are more readily identified crystallographically through their anomalous diffraction using softer X-rays. The protein conformation is very similar to that of the enzyme without CsCl in acetonitrile, both for the previously reported ( 1SCB ) and our own newly determined model. No fewer than 11 defined sites for Cs(+) cations and 8 Cl(-) anions are identified around the protein molecule, although most of these have partial occupancy and may represent nonspecific binding sites. Two Cs(+) and two Cl(-) ions are close to the mouth of the active site cleft, where they may affect catalysis. In fact, cross-linked CsCl-treated subtilisin crystals transferred to acetonitrile show catalytic activity several fold higher than the reference crystals containing Na(+). Presoaking with another large cation, choline, also increases the enzyme activity. The active site appears only minimally sterically perturbed by the ion presence around it, so alternative activation mechanisms can be suggested: an electrostatic redistribution and/or a larger hydration sphere that enhances the protein domain.

PubMed: 20099851
DOI: 10.1021/JA908703C

Experimental method

X-RAY DIFFRACTION (2.24 Å)