Catalytic mechanisms of thymidylate synthases

Abstract

The relationship between protein structure, motions, and catalytic activity is an evolving perspective in enzymology. An interactive approach, where experimental and theoretical studies examine the same catalytic mechanism, is instrumental in addressing this issue. We combine various techniques, including steady state and pre-steady state kinetics, temperature dependence of kinetic isotope effects (KIEs), site-directed mutagenesis, Xray crystallography, and quantum mechanics/molecular mechanics (QM/MM) calculations, to study the catalytic mechanisms of thymidylate synthase (TSase). Since TSase catalyzes the last step of the sole intracellular de novo synthesis of thymidylate (i.e. the DNA base T), it is a common target for antibiotic and anticancer drugs. The proposed catalytic mechanism for TSase comprises a series of bond cleavages and formations including activation of two C-H bonds: a rate-limiting C-H!C hydride transfer and a faster C-H!O proton transfer. This provides an excellent model system to examine the structural and dynamic effects of the enzyme on different C-H cleavage steps in the same catalyzed reaction. Our experiments found that the KIE on the hydride transfer is temperature independent while the KIE on the proton transfer is temperature dependent, implying the protein environment is better organized for H-tunneling in the former. Our QM/MM calculations revealed that the hydride transfer has a transition state (TS) that is invariable with temperature while the proton transfer has multiple subsets of TS structures, which corroborates with our experimental results. The calculations also suggest that collective protein motions rearrange the network of H-bonds to accompany structural changes in the ligands during and between chemical transformations. These computational results not only illustrate functionalities of specific protein residues that reconcile many previous experimental observations, but also provide guidance for future experiments to verify the proposed mechanisms. In addition, we conducted experiments to examine the importance of long-range interactions in TSase-catalyzed reaction, using Quinn, Prof. Claudio Margulis, Prof. Sara Mason, Prof. Lei Geng, and Prof. Alexei Tivanski. I have also received enormous help from the front office (Sharon Robertson), the Chem Store (Tim Koon and Andy Lynch), the mechanic shop (Frank Turner), the electronic shop (Mike Estenson), the IT center (David Sansbury, Jeff Miller, Jon Yates, Chris McGee, and Brenna Faler), the Chem Center (Janet Kugley and Jessica R. Alberhasky), and the Teaching labs (Earlene Erbe) et al. It would be impossible for me to accomplish anything in the past six years if you were not there to help, so thanks a lot to all of you. It is a wonderful department and I will miss you all.