Cross-talk between the Allosteric Effector-binding Sites in Mouse Ribonucleotide Reductase

Peter Reichard,Rolf Eliasson,Rolf Ingemarson,Lars Thelander

doi:10.1074/jbc.m005337200

Peter Reichard, Rolf Eliasson + Show 2 more

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https://doi.org/10.1074/jbc.m005337200

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Abstract

We compared the allosteric regulation and effector binding properties of wild type R1 protein and R1 protein with a mutation in the "activity site" (D57N) of mouse ribonucleotide reductase. Wild type R1 had two effector-binding sites per polypeptide chain: one site (activity site) for dATP and ATP, with dATP-inhibiting and ATP-stimulating catalytic activity; and a second site (specificity site) for dATP, ATP, dTTP, and dGTP, directing substrate specificity. Binding of dATP to the specificity site had a 20-fold higher affinity than to the activity site. In all these respects, mouse R1 resembles Escherichia coli R1. Results with D57N were complicated by the instability of the protein, but two major changes were apparent. First, enzyme activity was stimulated by both dATP and ATP, suggesting that D57N no longer distinguished between the two nucleotides. Second, the two binding sites for dATP both had the same low affinity for the nucleotide, similar to that of the activity site of wild type R1. Thus the mutation in the activity site had decreased the affinity for dATP at the specificity site, demonstrating the interaction between the two sites.

Highlights

All ribonucleotide reduction is allosterically controlled to ensure an appropriate supply of each of the four dNTPs required for DNA replication and repair [1,2,3]
Effector binding to the specificity site modulates the catalytic activity so that binding of ATP or dATP prepares the protein for the reduction of UDP or CDP, binding of dTTP prepares the protein for the reduction of GDP, and binding of dGTP prepares the protein for the reduction of ADP
Allosteric Regulation of the Catalytic Activity of Wild Type Mouse R1—In accordance with earlier results [17] concerning the calf thymus reductase, we found that the mouse enzyme reduced CDP, ADP, and GDP very poorly in the absence of allosteric effectors

Summary

Introduction

All ribonucleotide reduction is allosterically controlled to ensure an appropriate supply of each of the four dNTPs required for DNA replication and repair [1,2,3]. In the E. coli enzyme, this regulation of substrate specificity is only found when ATP binds to the activity site. The activity site regulates the overall activity of the enzyme so that binding of ATP stimulates enzyme activity, whereas binding of dATP inhibits it These effects are independent of the occupation at the specificity site. DGuo-L, provided an R1 protein insensitive to allosteric regulation by dGTP, suggesting a mutation in the specificity site [18]. R1 from the other cell line, dGuo-200 –1, was insensitive to inhibition by dATP, suggesting a mutation in the activity site [19]

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