Direct photoaffinity labeling of an allosteric site on subunit protein M1 of mouse ribonucleotide reductase by dTTP.

Abstract

The protein MI subunit of ribonucleotide reductase contains at least two allosteric nucleotide binding sites that control the capacity ofthe enzyme to reduce ribonucleotides to the deoxyribonucleotides required for DNA synthesis. Direct photoaffinity labeling of partially purified protein MI from mouse Tlymphoma (S49) cells was observed after UV irradiation in the presence of dTTP at 0C. The relative molar incorporation of nucleotide per subunit was 4-8%. Competition experiments showed that the dTTP was bound to an allosteric domain genetically and kinetically defined as the substrate specificity site of the enzyme. An altered protein MI isolated from a thymidine-resistant mutant cell line showed significantly decreased photoincorporation of dTTP, consistent with the fact that its CDP reductase activity is resistant to feedback inhibition by dTTP. Specific photolabeling of several other proteins with pyrimidine and purine nucleotides was also found, indicating the general usefulness of direct photoaffinity labeling in the study of enzymes involved in nucleotide and nucleic acid metabolism.The deoxyribonucleotides required for DNA synthesis are generated by reduction of the corresponding ribonucleotides in a reaction catalyzed by ribonucleotide reductase. A single enzyme, containing two nonidentical subunits, protein M1 and protein M2, reduces all four ribonucleoside diphosphate substrates and the regulation ofthe enzyme is under strict allosteric control (1). The overall activity of the nucleotide binding subunit, protein Ml, is influenced by the effectors ATP (stimulating) and dATP (inhibiting). The substrate specificity is regulated by either ATP (required for pyrimidine reduction), dTTP (required for GDP reduction), or dGTP (required for ADP reduction). This complex pattern ofregulation by effectors can be accounted for by postulating the existence of two types of allosteriebinding sites. One type binds ATP or dATP and governs the overall activity. The other type binds those NTP that govern the substrate specificity of the enzyme.Affinity labeling ofthe ribonucleotide reductase with specific ligands that affect the enzyme reaction would allow one to define and characterize molecularly the nucleotide binding sites of both normal and mutant enzymes. We have explored direct photoaffinity labeling by nucleotides, as recently described (2, 3). -Ribonucleotide reductase subunit protein Ml, partially purified from cultured mouse T-lymphoma cells (S49) (4), could be specifically photoaffinity labeled by using dTTP as the affinity ligand. Experiments with a mutant protein Ml that is unresponsive to regulation by dTTP (5) confirmed the specificity of the photoaffinity labeling technique and provided information about the mechanism of regulation of ribonucleotide reductase from deoxyribonucleoside-resistant mammaliancells. EXPERIMENTAL PROCEDURESThe protein Ml subunit of ribonucleotide reductase was partially purified from mouse T-lymphoma cells (S49) as described (4), using ammonium sulfate precipitation and a...

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