A KINETIC-STUDY ON THE INFLUENCE OF NUCLEOSIDE TRIPHOSPHATE EFFECTORSON SUBUNIT INTERACTION IN MOUSE RIBONUCLEOTIDE REDUCTASE

For enzymatic activity, mouse ribonucleotide reductase must form a het erodimeric complex composed of homodimeric RI and R2 proteins. Both su bstrate specificity and overall activity are regulated by the alloster ic effecters ATP, dATP, dTTP, and dGTP, which bind to two different si tes found on R1, the activity site and the substrate specificity site. We have used biosensor technique to directly observe the effects of t hese nucleotides on R1/R2 interactions. In the absence of effecters, p ositive cooperativity was observed with a Hill coefficient of 1.8 and a K-D of 0.5 mu M. In the presence of dTTP or dGTP, there was no coope rativity and subunit interaction was observed at a much lower R1 conce ntration. The highest R1/R2 affinity was in the presence of dATP or AT P with K-DS of 0.05-0.1 mu M. In all experiments, the molar stoichiome try between the subunits was close to 1:1. Our data support a model wh ereby binding of any of the effecters to the substrate specificity sit e promotes formation of the R1 dimer, which we believe is prerequisite for binding to the R2 dimer. Additional binding of either ATP (a posi tive effector) or dATP (a negative effector) to the activity site furt her increases R1/R2 association. We propose that binding of ATP or dAT P to the activity site controls enzyme activity, not by changing the a ggregation 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.