A better understanding of how enzymes activate covalent bonds will be pursued via the investigation of a small enzyme that catalyzes a single C-H bond activation, and these studies will be extended to a larger enzyme that catalyzes a complex cascade of covalent bond activations within a single active site. The studies aim to reveal the nature of the chemical step (bond activation), and the role of the whole protein structure and dynamics in that process. The studies will illuminate the evolutionary progressions that enhance the bond activation despite the fact that the catalytic turnover is usually rate-limited by processes other than the chemical transformation.
Four specific aims are proposed:
Aim 1 will follow the nature of the chemical step along the natural evolution of dihydrofolate reductase (DHFR) from bacteria to human, and from DHFR toward dihydrobiopterin reductase (DHPR) by means of directed evolution.
Aim 2 will examine the role of active site residues in different chemical conversions catalyzed by the enzyme thymidylate synthase (TSase), and will test experimentally an alternative reaction mechanism proposed by calculations.
Aim 3 will study the relations between the chemical step and fast equilibrium dynamics (femtosecond-nanosecond) across the whole protein.
Aim 4 will induce a minimal perturbation of those fast dynamics by means of isotopically heavy proteins (Born-Oppenheimer enzymes), and will explore the resultant effects on the catalyzed chemical step. Such comprehensive studies will require a broad arsenal of experimental and theoretical tools including measurements and calculations of kinetic isotope effects (KIEs);protein crystallography and measurements of anisotropic B-factors from X-ray diffractions;NMR relaxation measurements, hybrid QM/MM calculations;vibrational spectroscopy (2D-IR);and directed evolution. Accordingly, the research team is composed of fours subcontractors, three other co-investigators, and the PI.
Both DHFR and TSase are targets for antibiotic and chemotherapeutic drugs. The proposed studies will expose otherwise hidden chemical steps thus providing new targets for inhibitors and drug design. Understanding of the evolution of new substrate specificity will reveal molecular and physical features in the evolution of drug resistance. The expected outcome of the proposed studies will also shed light on the question of whether protein dynamics should be considered in rational drug design and biomimetic catalyst design.
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|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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