Role of protein dynamics in the catalysis by 6-hydroxylmethyl-7,8-dihydropterin pyrophosphokinase: A combined NMR and molecular dynamics simulation study

Proteins are intrinsically dynamic and it is believed that the dynamics has functional roles. While it is still a debate whether the protein dynamics is important for the chemical step of enzymatic function, there is an increasing consensus that protein dynamics plays important roles in the substrate binding and product release.;6-Hydroxylmethyl-7,8-dihydropterin pyrophosphokinase (HPPK) catalyzes the pyrophosphate transfer from ATP to 6-hydroxylmethyl-7,8-dihydropterin (HP), a key step in folate biosynthetic pathway. Evidence has indicated that HPPK dynamics is important for its function. Atomic structures have been determined for nearly every stage of its catalytic cycle. Comparison of those structures clearly shows that HPPK goes though dramatic conformational changes during the catalytic cycle, especially in the three catalytic loop regions.;In this thesis, the HPPK dynamics along the first half of the catalytic cycle, substrate binding, was studied using Nuclear Magnetic Resonance (NMR) and molecular dynamics (MD) simulation. The importance of protein dynamics was addressed by studies on two mutant proteins: Q50A and N10A HPPK. Q50 and N10 are key residues in the hydrogen-bond network found in the x-ray crystal structure of HPPK ternary complex, which couple the three catalytic loops together.;My results show that ligand-free (apo) HPPK is highly dynamic on a timescales ranging from picosecond to second, and binding of the first substrate does not reduce the internal dynamics but rather enhances it moderately, especially in the catalytic loop region. HPPK dynamics is largely quenched upon the binding of the second substrate, however, some mobility remains. The remaining dynamics may help the optimization of the active site interaction. Both N10 and Q50 are important for connecting the three catalytic loops and the loop coupling is important for the binding of the second substrate HP and the full assembling and stabilization of the active center and catalysis. MD simulation studies show that the active site residues pre-sample the side-chain conformation for substrate binding even without substrates, indicating that HPPK follows a selected-fit mechanism. One possible HPPK conformational transition pathway during the substrate binding is identified through targeted MD simulation.