Studies on the Radical Enzymes Glutamate Mutase and Viperin.

Adenosylcobalamin (coenzyme B12) serves as a source of organic radicals that are generated by homolytic scission of the cobalt-carbon bond to form cob(II)alamin and a highly reactive 5'-deoxyadenosyl radical, which then catalyzes a variety of unusual rearrangement reactions. The mechanism by which adenosylcobalamin-dependent enzymes accelerate cobalt-carbon bond homolysis by approximately one trillion-fold remains unclear. To gain molecular-level insight into this process for glutamate mutase, detailed kinetic analysis was combined with molecular dynamics simulations to examine a series of active site point mutations. These mutations cause a progressive increase in the mean distance between the 5'-carbon of the adenosyl radical and the substrate's abstractable hydrogen. This distance (determined computationally) was found to inversely correlate with the logk of tritium exchange between the coenzyme and the substrate (determined experimentally). The enzymatic homolysis of adenosylcobalamin may also be susceptible to external magnetic fields. A magnetic field can modulate the rate at which the initially formed singlet radical pair (which can recombine to reform the Co-C bond) converts to the triplet state (which cannot recombine). Magnetic field effect studies on glutamate mutase suggest that the 5'-deoxyadenosyl radical produced by homolysis is rapidly siphoned off by reaction with the substrate. This is consistent with the idea that the coupling of Co-C homolysis to hydrogen abstraction contributes to the acceleration of Co-C bond homolysis observed in adenosylcobalamin-dependent enzymes. Similar to adenosylcobalamin enzymes, radical SAM enzymes use an enzyme-bound [4Fe-4S] cluster to reductively cleave S-adenosyl-L-methionine (SAM) and generate a 5'-deoxyadenosyl radical that is then used to catalyze chemically-difficult transformations. Viperin, a newly identified radical SAM enzyme, is an interferon-stimulated gene that directs antiviral activity against a number of pathogens that infect eukaryotic cells. To gain insight into the mechanism by which viperin regulates viral replication, its interaction with farnesyl pyrophosphate synthase (the best-established target of viperin) was studied to elucidate the novel role radical SAM chemistry plays in the innate immune response. This study provides preliminary data that suggests viperin reduces cellular levels of farnesyl pyrophosphate synthase by approximately 50 percent.PhDChemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/111388/1/gdroman_1.pd