Enzymes catalyze a remarkable variety of chemical reactions with extremely high rate enhancements and very selective substrate specificity. The research efforts in our laboratory are directed towards a more complete understanding of the fundamental principles involved in enzyme-catalyzed chemistry and the dependence on protein structure. The pursuit of this information will provide the framework for the rational and combinatorial redesign of these complex molecules in an effort to exploit and develop the properties of enzyme active sites for a variety of chemical, biological, and medicinal uses. The techniques that we are using to solve these problems include steady-state and stopped-flow kinetics, NMR and EPR spectroscopy, X-ray crystallography, and the synthesis of inhibitors and suicide substrates. We are also using recombinant DNA methods to construct new proteins with novel catalytic properties. These efforts are currently being directed to the reactions catalyzed by carbamoyl phosphate synthetase, phosphotriesterase, and dihydroorotase.
The phosphotriesterase enzyme catalyzes the detoxification of organophosphate insecticides. We recently discovered that the active site of this protein consists of a unique binuclear metal center. We are now investigating the structure and properties of this metal center as a research tool for the evolution of enzyme structure and function. Carbamoyl phosphate synthetase catalyzes the formation of the key precursor for the biosynthesis of arginine and pyrimidine nucleotides. The complex heterodimeric protein contains three independent active sites connected by an internal molecular tunnel of 100 Å in length. It now appears that this protein operates as a unique molecular machine where the unstable intermediates are transported from one active site to the next via the controlled diffusion through the intermolecular tunnel. Dihydroorotase (DHO) catalyzes the reversible hydrolysis of the amide bond within dihydroorotate. This metabolic intermediate is required for the biosynthesis of pyrimidine nucleotides. DHO contains a binuclear metal center and is related to phosphotriesterase via divergent evolution
P.-C. Tsai, N. Fox, A. N. Bigley, S. P. Harvey, D. P. Barondeau, and F. M. Raushel, "Enzymes for the Homeland Defense: Optimizing Phosphotriesterase for the Hydrolysis of Organophosphate Nerve Agents" Biochemistry 51, 6463-6475 (2012).
S. S. Kamat, H. J. Williams, and F. M. Raushel, "Intermediates in the Transformation of Phosphonates to Phosphate in Bacteria" Nature 480, 570-573 (2011).
F. M. Raushel, "Catalytic Detoxification" Nature, 469, 310-311 (2011).
A. M. Goble, H. Fan, A. Sali, and F. M. Raushel, "The Discovery of a Cytokinin Deaminase" ACS Chemical Biology, 6, 1036-1040 (2011).
S. Kamat, H. Fan, J. M. Sauder, S. K. Burley, B. K. Shoichet, A. Sali, and F. M. Raushel, "Enzymatic Deamination of the Epigenetic Base N-6-Methyladenine" Journal of the American Chemical Society, 133, 2080-2083 (2011).
J. Hermann, R. Marti-Arbona, E. Fedorov, A. Fedorov, S. Almo, B. K. Shoichet, and F. M. Raushel, "Structure-Based Activity Prediction for an Enzyme of Unknown Function" Nature, 448, 775-779 (2007).
R. S. Hall, A. A. Fedorov, R. Marti-Arbona, E. V. Fedorov, P. Kolb, J. M. Sauder, S. K. Burley, B. K. Shoichet, S. C. Almo, and F. M. Raushel, "The Hunt for 8-Oxoguanine Deaminase" Journal of the American Chemical Society 132, 1762-1763 (2010).
D. S. Hitchcock, A. A. Fedorov, E. V. Fedorov, L. Dangott, S. C. Almo, and F. M. Raushel, "Rescue of the Orphan Enzyme Isoguanine Deaminase", Biochemistry 50, 5555-5557 (2011).