Thermococcus IPPase molecular mechanism elucidation
Figure 1. Comparisons of the active site among different ligation states. The X-ray crystal structures of different ligation states are compared. The conserved residues are shown in the stick model, the bound calcium ion(s) are shown as green spheres and water molecules are shown in red spheres.
Figure 2. Molecular dynamics outcome comparisons between calcium bound and magnecium bound TtIPPase. the location of relatively felxible reagion were differnt relative to its location to the active site.
Identifying the roles of water in enzymatic reaction and its thermal stability
The focus of this research is to decipher mechanism of inorganic pyrophosphatase from Thermococcus thioreducens (TtIPPase; E.C.number 126.96.36.199). This enzyme hydrolyzes pyrophosphate to two orthophosphates, and this highly exergonic reaction helps driving unfavorable biochemical reactions in the cells. Due to its crucial role, the active site residues are conserved among different domains of life. However, the role of each residue is not well-studied, and this conservative nature rises the question of what factors contributes to TtIPPase thermal stability.
Understanding how the presence of calcium contribute to the reaction inhibition
In addition, TtIPPase hydrolysis requires magnesium as a co-factor while the presence of calcium inhibits the reaction. As shown in the Figure 1, X-ray crystallographic structures highlight location changes of the calcium ions at the active site among different ligation states. Such orientation could be different in case of magnesium. However, what factors make two ions contribute to reaction outcome are not well-known.
In order to answer those questions, this research effort aims to combine all information from protein biochemistry experiments (carrying out site-directed mutagenesis, determination of those structures by both X-ray and Neutron crystallography and comparison of their kinetic parameters) and molecular dynamics simulation.