The long-term objective of the proposed research is to better understand how enzymes activate stable covalent bonds. A mechanistic investigation of C-H bond activation in alternative dihydrofolate reductases (DHFRs) and thymidylate synthase (TSase) will be conducted. These enzymes play crucial roles in DNA biosynthesis and thus serve as targets for antibiotic and chemotherapeutic drugs. They are also model systems used to address fundamental issues in enzymology, such as the role of protein dynamics and environmentally coupled quantum mechanical hydrogen tunneling in bond activation.
Aim 1 : The FolA encoded chromosomal DHFR (cDHFR) is a small protein that catalyzes a single chemical transformation and has been studied extensively during the previous grant period. Methods were developed to study the physical nature of the C-H-C transfer. It has been demonstrated that the cDHFR reaction coordinate is perfectly arranged for H-tunneling, and that mutants far from the active site can synergistically disturb the H-transfer process. These findings suggested a network of coupled motions across the enzyme that enhance the catalyzed reaction. The proposed studies will extend these studies to compare the effects of different protein scaffolds, dynamics, and reactants orientation on the physical nature of the C-H-C transfer.
Aim 2 : TSase is larger than cDHFR and catalyzes the making and breaking of multiple covalent bonds. We will examine different C-H activation steps in the complex TSase catalyzed reaction. These studies will include the examination of kinetic and dynamic effects of controlled active site mutations on different H- transfer steps. We will also examine the effect of altered networks of coupled motions on kinetics and dynamics using mutations distal to the active site. The findings from kinetic methods that can expose the nature of specific H-transfer steps will be correlated to measurements of protein dynamics, to assess the role of motions in enhancing C-H bond activation, in both fast and rate-limiting chemical steps.

Public Health Relevance

Two families of enzymes that are essential for DNA biosynthesis, and hence targets for antibiotic and chemotherapeutic drugs, will be studied. The investigation aims for a better understanding of how the dynamics of enzymes affect the chemistry they catalyze. The potential impact of including protein dynamics in drug design is far-reaching, and may boost practice of rational design in a field dominated by combinatorial approaches.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Macromolecular Structure and Function E Study Section (MSFE)
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Anderson, Vernon
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University of Iowa
Schools of Arts and Sciences
Iowa City
United States
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Klinman, Judith P; Kohen, Amnon (2014) Evolutionary aspects of enzyme dynamics. J Biol Chem 289:30205-12
Singh, Priyanka; Sen, Arundhuti; Francis, Kevin et al. (2014) Extension and limits of the network of coupled motions correlated to hydride transfer in dihydrofolate reductase. J Am Chem Soc 136:2575-82
Wang, Zhen; Singh, Priyanka; Czekster, Clarissa M et al. (2014) Protein mass-modulated effects in the catalytic mechanism of dihydrofolate reductase: beyond promoting vibrations. J Am Chem Soc 136:8333-41
Islam, Zahidul; Strutzenberg, Timothy S; Gurevic, Ilya et al. (2014) Concerted versus stepwise mechanism in thymidylate synthase. J Am Chem Soc 136:9850-3
Mishanina, Tatiana V; Corcoran, John M; Kohen, Amnon (2014) Substrate activation in flavin-dependent thymidylate synthase. J Am Chem Soc 136:10597-600
Francis, Kevin; Kohen, Amnon (2014) Protein motions and the activation of the CH bond catalyzed by dihydrofolate reductase. Curr Opin Chem Biol 21:19-24
Roston, Daniel; Kohen, Amnon (2014) Stereospecific multiple isotopic labeling of benzyl alcohol. J Labelled Comp Radiopharm 57:75-7
Roston, Daniel; Kohen, Amnon; Doron, Dvir et al. (2014) Simulations of remote mutants of dihydrofolate reductase reveal the nature of a network of residues coupled to hydride transfer. J Comput Chem 35:1411-7
Roston, Daniel; Islam, Zahidul; Kohen, Amnon (2014) Kinetic isotope effects as a probe of hydrogen transfers to and from common enzymatic cofactors. Arch Biochem Biophys 544:96-104
Wang, Zhen; Sapienza, Paul J; Abeysinghe, Thelma et al. (2013) Mg2+ binds to the surface of thymidylate synthase and affects hydride transfer at the interior active site. J Am Chem Soc 135:7583-92

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