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 and ATP, thereby providing balanced dNTP pools. We have characterized the allosteric 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 effectors for CTP reduction, dGTP is a positive effector for ATP reduction, and dTTP is a positive effector for GTP reduction. dATP is not a general negative allosteric effector. These effects are similar to the allosteric regulation of class Ib and class 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 coli and from Lactococcus lactis. The relative rate of reduction of the four substrates was measured simultaneously in a mixed-substrate assay, which mimics the physiological situation and illustrates the interplay between the different effectors in vivo. Surprisingly, we did not observe any significant UTP reduction under the conditions used. Balancing of the pyrimidine deoxyribonucleotide pools may be achieved via the dCMP deaminase and dCMP hydroxymethylase pathways.
Andersson
J
J
Department of Molecular Biology, Stockholm University, SE-10691 Stockholm, Sweden.
Westman
M
M
Hofer
A
A
Sjoberg
B M
BM
D000255:Adenosine Triphosphate / Q000378:metabolism; D000494:Allosteric Regulation; D000495:Allosteric Site; D017122:Bacteriophage T4 / Q000737:chemistry*; D001667:Binding, Competitive; D003570:Cytidine Triphosphate / Q000378:metabolism; D003838:Deoxyadenine Nucleotides / Q000378:metabolism; D003845:Deoxycytosine Nucleotides / Q000378:metabolism; D003848:Deoxyguanine Nucleotides / Q000378:metabolism; D003856:Deoxyuracil Nucleotides / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D007700:Kinetics; D009711:Nucleotides / Q000378:metabolism; D012264:Ribonucleotide Reductases / Q000737:chemistry* / Q000378:metabolism*; D013379:Substrate Specificity; D013997:Time Factors; D014544:Uridine Triphosphate / Q000378:metabolism
D003838:Deoxyadenine Nucleotides; D003845:Deoxycytosine Nucleotides; D003848:Deoxyguanine Nucleotides; D003856:Deoxyuracil Nucleotides; D009711:Nucleotides; D003570:Cytidine Triphosphate; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D012264:Ribonucleotide Reductases; C101141:anaerobic ribonucleotide reductase; D014544:Uridine Triphosphate