Ab-initio simulations of the catalytic mechanism in protein kinases
EMSL Project ID
7191
Abstract
Protein kinase enzymes play an important role in controlling cellular signaling events by catalyzing a phosphorylation reaction in which the gamma-phosphate from an adsorbed ATP in the catalytic pocket of the enzyme is transferred to a substrate protein. Despite many structural and kinetic measurements, there is considerable controversy regarding the chemical mechanism of the phosphorylation and the roles of the highly conserved active site residues in facilitating the reaction. In the proposed program, these issues will be addressed using a combined quantum mechancs/molecular mechanics (QMMM) approach, as implemented in the NWChem computational chemistry package. Our study will be concentrated on two members of the protein kinase family?cAMP dependent serine protein kinase and insulin receptor tyrosine kinase. Using available x-ray data (1ATP, 1IR3) we will construct comprehensive models of the active sites of these enzymes. These models will contain all the key residues believed to be important to the phosphorylation process. The properties of the putative active sites will be studied using ab-initio calculations based on density functional theory. Effects of the surrounding protein will be treated using a molecular mechanics approach. Our earlier quantum-mechanical simulations (Valiev, M.; Kawai, R.; Adams, J. A.; Weare, J. H.; JACS, 125, 9926, 2003) have shown that the reaction mechanism in cAPK serine kinase is predominantly dissociative. We have also demonstrated that Asp166 plays an important role in the reaction process as a proton acceptor late in the reaction process. This is consistent with experimental data. In this project, these simulations will be extended to include a more complete description of the cAPK kinase active site, including the effects of the protein environment. To understand the differences between serine and tyrosine kinases, a similar study will be carried out for 1IR3 insulin receptor tyrosine kinase. These calculations will help us to identify the role of another important residue, Lys168. Replacement of Lys168 by Ala in serine yeast kinase leads to 1600-fold decrease in catalytic efficiency. However, unlike the Asp166, which is conserved in all kinases, Lys186 is replaced by Arg in tyrosine kinases. Comparative studies of serine and tyrosine kinases reaction mechanisms will contribute to the understanding of the role of this residue.
To provide a comprehensive and reliable view of the reaction process, our simulations will require a quantum-mechanical active site model that contains up to 400 atoms. To obtain the required accuracy, a converged ab-initio (B3LYP) treatment using a high quality basis set (thousands of basis functions) will be necessary. This presents a formidable computational problem that can only be addressed using massively parallel hardware and software.
Project Details
Project type
Capability Research
Start Date
2004-03-11
End Date
2006-04-04
Status
Closed
Released Data Link
Team
Principal Investigator
Team Members