The hallmarks of enzyme catalysis are its exquisite specificity and highly accelerated reaction rates. In order to understand the fundamental processes that endow these features to enzymatic catalysis, our group, over last several years, has used dihydrofolate reductase (DHFR) as a model enzyme. The reasons behind using DHFR are manifold, including the ease of obtaining the enzyme in large quantities and substantial literature on its structure and reaction kinetics. DHFR is also an important target of clinical intervention. Methotrexate, an important anticancer agent, and trimethoprim, an antimicrobial, exhibit their therapeutic effect primarily by inhibition of DHFR.
DHFR catalyzes the NADPH-dependent reduction of 7,8-dihydrofolate (H2F) to 5,6,7,8-tetrahydrofolate (H4F) and is necessary for maintaining intracellular levels of H4F, an essential cofactor in the synthetic pathway of purines, thymidylate and several amino acids.
Although, a variety of factors are responsible for the enormous catalytic power of enzymes, we are particularly interested in studying the role of protein dynamics as it relates to catalysis. In order to investigate the role of protein dynamics, we probe the communication between distal sites by performing double and higher order mutant cycle experiments. Pre-steady state kinetic studies on these mutants provide energies of coupling between different parts of the enzyme and contribution of these long-range, dynamic interactions towards catalysis.
In a series of further studies, using the knowledge of coupled interactions between the distal residues, we are examining whether the conformational changes contribute towards enhancing the reaction rate, in addition to facilitating the precise alignment of the reactive groups for the chemistry to ensue. To address such questions, ensemble kinetics and single molecule TIRF microscopy experiments (in collaboration with Prof. Gordon Hammes) are being performed. In our experimental setup, fluorescence quencher/fluorescent probe dye molecule pairs are covalently attached to chosen residue pairs in DHFR. In stopped-flow ensemble experiments, we follow fluorescence changes of the donor probe during the reaction catalyzed by DHFR. These experiments allow us to follow the changes between the labeled residues during catalysis and obtain the rates of specific conformational changes. Moreover, the fluctuation in fluorescence intensity of single molecules of DHFR is recorded during catalysis at equilibrium conditions to pull apart the individual motions hidden under the unsynchronized ensemble measurements. The long term goal of these studies is to experimentally develop an accurate understanding of DHFR dynamics during catalysis in three dimensions and to determine the specific roles served by these fluctuations.
In parallel, the network of coupled promoting motions in DHFR is being theoretically characterized using mixed quantum/classical molecular dynamic simulations of hydride transfer in collaboration with the lab of Prof. Sharon Hammes Schiffer. These hybrid simulations, in combination with the rank correlation analysis, also enable delineation of the individual residues involved in coupled motions along the reaction coordinate.
In another effort, we have mapped the network of interacting residues in DHFR using the Statistical Coupling Analysis (in collaboration with Prof. Rama Ranganathan). This information is being used to design artificial protein sequences that would show DHFR activity. In addition, this information is being used to locate potential sites for grafting modules to relay allosteric signals to control the DHFR activity.
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Representative
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Important
Reviews:
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Hammes-Schiffer, S. and Benkovic, S. J. (2006) Relating Protein Motion to Catalysis, Annu. Rev. Biochem. 75, 519-541. |
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