A kinetic study on the influence of nucleoside triphosphate effectors on subunit interaction in mouse ribonucleotide reductase.
For enzymatic activity, mouse ribonucleotide reductase must form a heterodimeric complex composed of homodimeric R1 and R2 proteins. Both substrate specificity and overall activity are regulated by the allosteric effectors ATP, dATP, dTTP, and dGTP, which bind to two different sites found on R1, the activity site and the substrate specificity site. We have used biosensor technique to directly observe the effects of these nucleotides on R1/R2 interactions. In the absence of effectors, positive cooperativity was observed with a Hill coefficient of 1.8 and a KD of 0.5 microM. In the presence of dTTP or dGTP, there was no cooperativity and subunit interaction was observed at a much lower R1 concentration. The highest R1/R2 affinity was in the presence of dATP or ATP with KDs of 0.05-0.1 microM. In all experiments, the molar stoichiometry between the subunits was close to 1:1. Our data support a model whereby binding of any of the effectors to the substrate specificity site promotes formation of the R1 dimer, which we believe is prerequisite for binding to the R2 dimer. Additional binding of either ATP (a positive effector) or dATP (a negative effector) to the activity site further increases R1/R2 association. We propose that binding of ATP or dATP to the activity site controls enzyme activity, not by changing the aggregation state of the R1/R2 proteins as proposed earlier, but rather by locally influencing the long range electron transport between the catalytic site of R1 and the tyrosyl free radical of R2.
Ingemarson
R
R
Department of Medical Biochemistry and Biophysics, Umeå University, Sweden. roling@panther.cmb.umu.se
Thelander
L
L
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000494:Allosteric Regulation; D000818:Animals; D003838:Deoxyadenine Nucleotides / Q000378:metabolism*; D003848:Deoxyguanine Nucleotides / Q000378:metabolism*; D007700:Kinetics; D051379:Mice; D011485:Protein Binding; D012264:Ribonucleotide Reductases / Q000378:metabolism*; D013942:Thymine Nucleotides / Q000378:metabolism*
D003838:Deoxyadenine Nucleotides; D003848:Deoxyguanine Nucleotides; D013942:Thymine Nucleotides; C029603:deoxyguanosine triphosphate; D000255:Adenosine Triphosphate; D012264:Ribonucleotide Reductases; C024157:thymidine 5'-triphosphate