Gira Bhabha
Published: 2011
Total Pages: 532
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Conformational dynamics are important for a variety of biological functions, including enzyme catalysis. Many questions remain as to the role of protein dynamics in enzyme catalysis, and these questions are best addressed in model systems for which detailed kinetic, biological and biophysical data can be obtained. Dihydrofolate reductase (DHFR) is an enzyme present in almost all cell types, and catalyzes the stereospecific reduction of dihydrofolate (DHF) to tetrahydrofolate (THF) using NADPH as a cofactor. THF is a precursor for thymidylate synthesis and is, therefore, required for cell proliferation. Much biochemical and structural work has shed light on various aspects of E. coli DHFR (ecDHFR), making it a paradigm for understanding enzyme mechanism. We have studied the protein conformational motions present in human DHFR (hDHFR), and find that despite a high level of structural conservation between hDHFR and ecDHFR, the dynamic mechanisms of the two enzymes are quite different. While E. coli DHFR employs an extensive loop motion to facilitate ligand flux, its human counterpart has evolved a more subtle and sophisticated twisting-hinge motion to the same end. Based on the differences in flexibility and motion in these two enzymes, we designed a mutant of ecDHFR, which abolishes the active site dynamics without perturbing the protein structure or preorganization of the active site configuration. This mutant allowed us to assess the role of dynamics in ecDHFR catalysis, and we found that the millisecond timescale dynamics play a critical role in the chemical step of catalysis as well as in ligand flux for ecDHFR. Combining these detailed structural studies on ecDHFR and hDHFR with bioinformatic analyses, mutagenesis, x-ray and NMR data, we are able to gain insights into the evolution of DHFR dynamic mechanisms at an atomic level.