X-ray studies of metalloenzyme regulation and catalysis

Massachusetts Institute Of Technology, Cambridge MA

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Abstract

The broad, long term goal of this research is to elucidate the role of conformational change in catalysis and enzyme regulation. Our research will emphasize the allosteric regulation and inter- and intramolecular communication of riboucleotide reductases (RNRs). These amazing enzymes utilize organic radical-based chemistry to convert nucleotides to deoxynucleotides. Allosteric regulation of RNR enzymes serves to maintain an appropriate supply of each of four deoxynucleotides (dNTPs) needed for DNA replication and repair. As a result of these cellular activities, RNRs are actively pursued as targets for antibacterial, antiviral, and antitumor therapies. Gemcitabine (2',2'-difluoro-2'-deoxycytidine), a mechanism-based inhibitor of RNRs, was recently approved by the FDA for treatment of pancreatic cancer and demonstrates the medical importance of these enzymes. The proposed research is intended to identify the conformational changes involved in RNR re-reduction by thioredoxin/thioredoxin reductase/NADPH, to identify the role of conformational change in the allosteric regulation of radical formation, and to identify the structural basis for allosteric regulation of enzyme specificity. These goals will be achieved by crystallographic analysis of the least complex members of the ribonucleotide reductase family, the B12-dependent class II enzymes. We will complement structural data with biochemical characterization and single crystal spectroscopic techniques.