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.

Public Health Relevance

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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Postdoctoral Individual National Research Service Award (F32)
Project #
5F32GM087034-03
Application #
8119447
Study Section
Special Emphasis Panel (ZRG1-F04B-L (20))
Program Officer
Fabian, Miles
Project Start
2009-08-01
Project End
2012-07-31
Budget Start
2011-08-01
Budget End
2012-07-31
Support Year
3
Fiscal Year
2011
Total Cost
$53,042
Indirect Cost
Name
Massachusetts Institute of Technology
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
001425594
City
Cambridge
State
MA
Country
United States
Zip Code
02139
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