Allosteric regulation of the class III anaerobic ribonucleotide reductase from bacteriophage T4

Ribonucleotide reductase (RNR) is an essential enzyme in all organisms, It provides precursors for DNA synthesis by reducing all four ribonucleotides to deoxyribonucleotides, The overall activity and the substrate specificity of RNR are allosterically regulated by deoxyribonucleoside triphosphates a nd ATP, thereby providing balanced dNTP pools, We have characterized the al losteric regulation of the class III RNR from bacteriophage T4. Our results show that the T4 enzyme has a single type of allosteric site to which dGTP , dTTP, dATP, and ATP bind competitively. The dissociation constants are in the micromolar range, except for ATP, which has a dissociation constant in the millimolar range. ATP and dATP are positive effecters for CTP reductio n, dGTP is a positive effector for ATP reduction, and dTTP is a positive ef fector for GTP reduction. dATP is not a general negative allosteric effecto r, These effects are similar to the allosteric regulation of class Ib and c lass II RNRs, and to the class Ia RNR of bacteriophage T4, but differ from that of the class III RNRs from the host bacterium Escherichia coil and fro m Lactococcus lactis. The relative rate of reduction of the four substrates was measured simultan eously in a mixed-substrate assay, which mimics the physiological situation and illustrates the interplay between the different effecters in vivo Surp risingly, we did not observe any significant UTP reduction under the condit ions used, Balancing of the pyrimidine deoxyribonucleotide pools may be ach ieved via the dCMP deaminase and dCMP hydroxymethylase pathways.