A Kinetic Study on the Influence of Nucleoside Triphosphate Effectors on Subunit Interaction in Mouse Ribonucleotide Reductase

Abstract

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.

Mutant R1 Proteins from Escherichia coli Class Ia Ribonucleotide Reductase with Altered Responses to dATP Inhibition

“…This theory has been discussed in several reports (3,6,22). In our preliminary experiments, we found that high concentrations of dATP promote the formation of a tight complex.…”

Yeast DNA Damage-inducible Rnr3 Has a Very Low Catalytic Activity Strongly Stimulated after the Formation of a Cross-talking Rnr1/Rnr3 Complex

“…Cross-talk between the Allosteric Activity Site in Rnr1-C428A and the Catalytic Site in Rnr3-It was shown earlier for the mouse RNR complex that allosteric effectors strongly influence subunit interaction (28). Reduction of CDP by yeast RNR containing the Rnr1 was stimulated by ATP with maximal activity around 5 mM ATP (Fig.…”

Enhanced subunit interactions with gemcitabine-5′-diphosphate inhibit ribonucleotide reductases

“…Current evidence suggests that there is one Y • and 2 di-iron clusters/␤2 (19,20). The interactions between ␣ and ␤ are weak (K d of 0.2 M) in the absence of nucleotide (21). In prokaryotic systems ␣ and ␤ are both homodimers.…”

“…Recent gas-phase electrophoretic-mobility macromolecule analysis studies have suggested that the mouse quaternary structure is ␣6␤2 (24). Studying subunit interactions has been difficult by conventional methods because of ␣'s nucleotide-dependent aggregation state and the weak binding of ␣ and ␤, which is also nucleotide-dependent (21). Determination of apparent molecular masses of the active RNR complexes requires nucleotides in the analysis buffer, and thus protein detection by UV spectroscopy is obscured (18,23).…”

“…The instability of the ␤2 radical requires that a control in the absence of F 2 CDP be carried out and used to correct the data observed in its presence. The weak interaction between ␣ and ␤ of RNR allow assays of the subunit activity individually (21,32). Thus, the inactivation mixture was assayed for activity of ␣ (␤) in the presence of a 7-fold excess of ␤ (␣).…”