4GEV

E. coli thymidylate synthase Y209W variant in complex with substrate and a cofactor analog

Replaces:  2G8M

Summary for 4GEV

• Entry DOI: 10.2210/pdb4gev/pdb
• Related: 2G86 2G89 2G8A 2G8D 2G8O 2G8X
• Descriptor: Thymidylate synthase, 2'-deoxy-5'-uridylic acid, 10-PROPARGYL-5,8-DIDEAZAFOLIC ACID, ... (4 entities in total)
• Functional Keywords: beta sheet, alpha/beta protein, methylase, transferase
• Biological source: Escherichia coli
• Cellular location: Cytoplasm : P0A884
• Total number of polymer chains: 2
• Total formula weight: 62740.73

Authors

Newby, Z.,Lee, T.T.,Finer-Moore, J.,Stroud, R.M. (deposition date: 2012-08-02, release date: 2012-08-29, Last modification date: 2024-11-27)

Primary citation

Wang, Z.,Abeysinghe, T.,Finer-Moore, J.S.,Stroud, R.M.,Kohen, A.
A remote mutation affects the hydride transfer by disrupting concerted protein motions in thymidylate synthase.
J.Am.Chem.Soc., 134:17722-17730, 2012

PubMed Abstract: The role of protein flexibility in enzyme-catalyzed activation of chemical bonds is an evolving perspective in enzymology. Here we examine the role of protein motions in the hydride transfer reaction catalyzed by thymidylate synthase (TSase). Being remote from the chemical reaction site, the Y209W mutation of Escherichia coli TSase significantly reduces the protein activity, despite the remarkable similarity between the crystal structures of the wild-type and mutant enzymes with ligands representing their Michaelis complexes. The most conspicuous difference between these two crystal structures is in the anisotropic B-factors, which indicate disruption of the correlated atomic vibrations of protein residues in the mutant. This dynamically altered mutant allows a variety of small thiols to compete for the reaction intermediate that precedes the hydride transfer, indicating disruption of motions that preorganize the protein environment for this chemical step. Although the mutation causes higher enthalpy of activation of the hydride transfer, it only shows a small effect on the temperature dependence of the intrinsic KIE, suggesting marginal changes in the geometry and dynamics of the H-donor and -acceptor at the tunneling ready state. These observations suggest that the mutation disrupts the concerted motions that bring the H-donor and -acceptor together during the pre- and re-organization of the protein environment. The integrated structural and kinetic data allow us to probe the impact of protein motions on different time scales of the hydride transfer reaction within a complex enzymatic mechanism.

PubMed: 23034004
DOI: 10.1021/ja307859m

Experimental method

X-RAY DIFFRACTION (1.3 Å)