This research will investigate the kinetics and inhibition of radical initiation in Ribonucleotide Reductase (RNR). Artificial fluorotyrosine amino acids will be installed along the pathway, and the whole enzyme will be reconstituted in vitro. For the first study, photooxidants will be incorporated to jump start the radical propagation pathway by photoexcitation/oxidation. Radical propagation will be measured by the changes in transient absorption of unnatural and natural amino acid radicals. In the second study, photolabile protecting groups (photocages) will be incorporated onto a fluorotyrosine, and the holoenzyme will again be reconstituted. Here, photoexcitation will generate a redox-active phenolate, which should induce radical propagation along the entire pathway of radical initiation. From these experiments we will be able to derive the kinetic parameters for specific amino acids along the radical initiation pathway, as well as deduce the kinetics of the overall radical transport process in the assembled enzyme system. Third, once the kinetics for each system have been determined, it will be possible to probe the effect of radical scavengers such as hydroxyurea on the process of radical initiation. RNR is obligate forth production of deoxynucleosldes in prokaryotes and eukaryotes, and has long been a target for inhibition in disease states requiring aberrant or unnatural DNA production, including HIV-1 and cancers. The kinetic information obtained for the enzyme, and the effect on the enzyme by hydroxyurea, will allow the development of improved therapeutics targeting the radical initiation pathway of RNR.
This research will study the way in which an enzyme, ribonucleotide reductase, can be targeted with therapeutic drug molecules. Knowledge learned from these studies may allow better drugs to be designed that prevent cancers and HIV from exploiting the enzyme as they grow and replicate.
| Song, David Y; Pizano, Arturo A; Holder, Patrick G et al. (2015) Direct Interfacial Y731 Oxidation in ?2 by a Photo?2 Subunit of E. coli Class Ia Ribonucleotide Reductase. Chem Sci 6:4519-4524 |
| Pizano, Arturo A; Olshansky, Lisa; Holder, Patrick G et al. (2013) Modulation of Y356 photooxidation in E. coli class Ia ribonucleotide reductase by Y731 across the ?2:?2 interface. J Am Chem Soc 135:13250-3 |
| Pizano, Arturo A; Lutterman, Daniel A; Holder, Patrick G et al. (2012) Photo-ribonucleotide reductase ?2 by selective cysteine labeling with a radical phototrigger. Proc Natl Acad Sci U S A 109:39-43 |
| Holder, Patrick G; Pizano, Arturo A; Anderson, Bryce L et al. (2012) Deciphering radical transport in the large subunit of class I ribonucleotide reductase. J Am Chem Soc 134:1172-80 |
