Cross-talk between the allosteric effector-binding sites in mouse ribonucleotide reductase.
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.
Reichard
P
P
Department of Biochemistry 1, Medical Nobel Institute, MBB, Karolinska Institute, SE-17177 Stockholm, Sweden.
Eliasson
R
R
Ingemarson
R
R
Thelander
L
L
D000255:Adenosine Triphosphate / Q000378:metabolism; D000494:Allosteric Regulation; D000495:Allosteric Site; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002384:Catalysis; D003838:Deoxyadenine Nucleotides / Q000494:pharmacology; D003854:Deoxyribonucleotides / Q000378:metabolism / Q000494:pharmacology; D007700:Kinetics; D051379:Mice; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012264:Ribonucleotide Reductases / Q000737:chemistry* / Q000378:metabolism*
D003838:Deoxyadenine Nucleotides; D003854:Deoxyribonucleotides; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D012264:Ribonucleotide Reductases; C026600:2'-deoxyadenosine triphosphate