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 funding 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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM065368-08
Application #
8089560
Study Section
Macromolecular Structure and Function E Study Section (MSFE)
Program Officer
Anderson, Vernon
Project Start
2002-07-01
Project End
2013-04-30
Budget Start
2011-05-01
Budget End
2012-04-30
Support Year
8
Fiscal Year
2011
Total Cost
$271,574
Indirect Cost
Name
University of Iowa
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
062761671
City
Iowa City
State
IA
Country
United States
Zip Code
52242
?widerek, Katarzyna; Arafet, Kemel; Kohen, Amnon et al. (2017) Benchmarking Quantum Mechanics/Molecular Mechanics (QM/MM) Methods on the Thymidylate Synthase-Catalyzed Hydride Transfer. J Chem Theory Comput 13:1375-1388
Guo, Qi; Gakhar, Lokesh; Wickersham, Kyle et al. (2016) Structural and Kinetic Studies of Formate Dehydrogenase from Candida boidinii. Biochemistry 55:2760-71
?widerek, Katarzyna; Moliner, Vicent (2016) Computational Studies of Candida Antarctica Lipase B to Test Its Capability as a Starting Point To Redesign New Diels-Alderases. J Phys Chem B 120:2053-70
Francis, Kevin; Sapienza, Paul J; Lee, Andrew L et al. (2016) The Effect of Protein Mass Modulation on Human Dihydrofolate Reductase. Biochemistry 55:1100-6
Singh, P; Islam, Z; Kohen, A (2016) Examinations of the Chemical Step in Enzyme Catalysis. Methods Enzymol 577:287-318
Abeysinghe, Thelma; Hong, Baoyu; Wang, Zhen et al. (2016) Preserved hydride transfer mechanism in evolutionarily divergent thymidylate synthases. Curr Top Biochem Res 17:19-30
Krzemi?ska, Agnieszka; Moliner, Vicent; ?widerek, Katarzyna (2016) Dynamic and Electrostatic Effects on the Reaction Catalyzed by HIV-1 Protease. J Am Chem Soc 138:16283-16298
Kholodar, Svetlana A; Kohen, Amnon (2016) Noncovalent Intermediate of Thymidylate Synthase: Fact or Fiction? J Am Chem Soc 138:8056-9
Sapienza, Paul J; Falk, Bradley T; Lee, Andrew L (2015) Bacterial Thymidylate Synthase Binds Two Molecules of Substrate and Cofactor without Cooperativity. J Am Chem Soc 137:14260-3
Singh, Priyanka; Abeysinghe, Thelma; Kohen, Amnon (2015) Linking protein motion to enzyme catalysis. Molecules 20:1192-209

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