Question

Class I ribonucleotide reductase is most active in the presence of high ATP concentrations as a hexamer of the (R1/R2) heterodimer.  Molar masses of the subunits are :R1: 84 kD and R2: 40kD.  Design an experiment to test whether ATP is indeed involved in hexamerization of ribonucleotide reductase.  You must sketch and explain the results of your experiment and include a control experiment for full credit!

Answer 1

Site-directed mutagenesis and functional assays confirm that hexamerization is a prerequisite for inhibition by dATP. Our data indicate a mechanism for regulating RR activity by dATP-induced oligomerization.

Ribonucleotide reductase (RR) is an αnβn (RR1–RR2) complex that maintains balanced dNTP pools by reducing NDPs to dNDPs.

RR1 is the catalytic subunit, and RR2 houses the free radical required for catalysis. RR is allosterically regulated by its activator ATP and its inhibitor dATP, which regulate RR activity by inducing oligomerization of RR1.

Here, we report the first X-ray structures of human RR1 bound to TTP alone, dATP alone, TTP–GDP, TTP–ATP, and TTP–dATP. These structures provide insights into the regulation of RR by ATP or dATP. At physiological dATP concentrations, RR1 forms inactive hexamers.

We determined the first X-ray structure of the RR1–dATP hexamer and used single-particle electron microscopy to visualize the α6–ββ′–dATP holo complex.

The relative ratios between the dNTPs are controlled by the allosteric specificity site (the s-site), where binding of ATP/dATP induces CDP/UDP reduction and dTTP and dGTP induce GDP and ADP, respectively.