This award will support work by Professor Justine Roth at Johns Hopkins University to examine a plant-derived fatty acid alpha-dioxygenase which is believed to be a member of a family of enzymes that utilize tyrosyl radicals for catalysis. The ability of protein-derived radicals to mediate selective C-H oxidation using molecular oxygen has not been understood. The project features a mechanistic study that will allow the kinetics and thermodynamics of fatty acid oxidation to be characterized. Measurements of deuterium kinetic isotope effects as a function of temperature and substrate C-H bond strength will reveal the nature of the reaction barrier, which in this case also controls enzyme turnover. The results will be analyzed using Marcus Theory to elucidate the origins of enzyme catalysis. Insights of this kind are fundamentally important in the rational engineering of biological and chemical catalysts as well as the applications of enzymes in the industrial production of commodity chemicals. A broader objective of this research is to expose students to the importance of oxidative phenomena in chemistry and biology. An educational enrichment program for Baltimore City high school students has been initiated in collaboration with the Teach for America Baltimore Corps. This program will expose students to higher education in the biological and physical sciences, specifically at Johns Hopkins University. The program will also cultivate opportunities for summer internships and research experiences within the P.I.'s laboratories.
Overview: Cyclooxygenase enzymes (COX-1 and COX-2), previously prostaglandin H synthases, are tyrosyl radical (Y•) utilizing hemoproteins in the nuclear membranes of mammalian cells. They function within almost every cell type, dually as a peroxidase and a dioxygenase. The peroxidase reactions consume anti-oxidants and certain neurotransmitters that reduce hydroperoxide compounds, which are intermediate products of the enzyme’s dioxygenase function. In these reactions, O2 combines with polyunsaturated fatty acids (PUFAs) to form prostanoids and other signaling molecules in humans. Recently, COX-2 was deemed unique in its ability to oxidize endogenous cannabinoids (ECs) in human subjects. Note: ECs are simple derivatives of PUFAs; i.e. condensation products of a fatty acid and ethanolamine or glycerol. The ECs bind to cannabinoid receptor site(s), named for a psychoactive substance found in cannabis plants. Exploiting control levels over EC stimulation is a new area in pharmaceutical chemistry being pursued for its promise in homeopathic medical treatments. These "cures" have implications for the general regulation of mood and in central and peripheral disturbances of the nervous system. The COX enzymes impact pain levels, hemostasis and inflammation throughout endothelial cells. COX-2 is believed to have specific immunologic and neurologic effects from COX-1 and suspected to cause cardiovascular events in a small fraction of human subjects. These observations indicate that enzymes mechanisms may differ! In all currently sponsored research efforts, COX-2 is assumed to react "by the same mechanism" as COX-1; this presumes that the enzymes well established structural relationship translates into a mechanistic relationship. The circulation of COX-2 specific pharmaceutical has been limited to one drug in the US population. Rational design of such drugs requires understanding an enzyme’s kinetic and mechanistic abilities under rigorously defined conditions. As described in this proposal, a combination of heavy and light isotopic techniques (together with density functional theory calculations) allow us to evaluate transition states in enzymes based on changes in bond force constants. Intellectual Merit: The subject of this study, COX-2, is of fundamental importance, from pharmaceutical design to translational medicine. Implementation of new medical treatments relies on exploiting enzyme inhibitors’ mechanistic abilities. The proposed research describes an "enzymological" interrogation of COX-2 in view of its implications for pharmacology and neuroscience. Although specific COX-2 inhibitors have been synthesized, only one is FDA approved and prescribed, rather than sold "over-the-counter", because of the potential for adverse cardiovascular effects in a fraction of the population. New biophysical (specifically biochemical) understanding is needed to conceive of useful pharmaceutical strategies. COX-2 is an important mechanistic target because of its promise in developing agonists for anti-cancer chemotherapies, prophylactic use of neuro-protectants and treatment of neuropsychiatrtic disorders (related to learning, appetite and addictive behaviors). Broader Impacts: Understanding redox enzyme catalysis relies on integration of biology, chemistry and physics. This important molecular biophysical approach highlights the intriguing possibility that mechanisms conducive to nuclear tunneling are intrinsic to an enzymes’ ability to catalyze reactions and that isotope effects can be used to relate critical experimental and theoretical findings. The inter-disciplinary science in this proposal should have a broad impact on pedagogy and medicine. In part, the findings will involve students at the high school level. The Bryn Mawr School, a STEM-intensive (all female) high school in Baltimore City has promoted research programs. Students interested in the P.I.’s research will be identified through the STEM coordinator.
