Efforts will continue to unravel the mechanism of and role of the cofactors in ribonucleotide reductases from E. coli, L. leichmannii and B. ammoniagenes. These three systems have been chosen as prototypes of the binuclear iron center tyrosylphenoxy radical, adenosylcobalamin and uncharacterized """"""""Mn"""""""" center, respectively unique cofactors responsible for this unusual reduction reaction, the rate determining step in DNA biosynthesis. The basic objectives with the E. coli reductase (RDPR) are: 1. To elucidate, using the method of site directed mutagenesis, the role of the recently discovered two sets of redox dithiols. 2. To use these enzyme mutants and alternate nucleotide analogs 2' FNDP and 2,2' diFNDPs as probes for the mechanism of substrate reduction. 3. To define the structure of the sugar moiety remaining and the sequence of the modified Bl peptide produced subsequent to inactivation of RDPR by N3UDP. 4. To use 3'-N3NDP, 02' 2'3'-epoxoNDP and 2'-SHNDPs as potential probes of radical chemistry in the reductase catalyzed reactions. 5. To examine the in vitro mechanism of assembly of the binuclear iron center and to define ligands bound to the iron center. The basic objectives with the L. leichmannii reductase (RTPR) are: 1. To isolate and sequence the gene for RTPR and to attempt to express the gene in E. coli. 2. To examine the interaction of isotopically labeled araN3ATP as a probe for the postulated protein radical. 3. To examine the inactivation of RTPR using isotopically labeled N3UTPs. 4. To examine the role of RTPR and AdoCbl during inactivation by specifically labeled araClATPs - as a model for AdoCb1 in the rearrangement reactions. The basic objectives with the B. ammoniagenes reductase are: 1. To isolate the protein and to obtain peptide sequence information. To use specifically isotopically labeled nucleotides to examine the similarities/differences between this protein and the E. coli and L. leichmannii reductases.
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