Abstract
We have used 8-azidoadenosine 5'-triphosphate (8-N3ATP) to investigate the nucleotide-binding sites on the NrdD subunit of the anaerobic ribonucleotide reductase from T4 phage. Saturation studies revealed two saturable sites for this photoaffinity analog of ATP. One site exhibited half-maximal saturation at approximately 5 microM [gamma-32P]8-N3ATP, whereas the other site required 45 microM. To localize the sites of photoinsertion, photolabeled peptides from tryptic and chymotryptic digests were isolated by immobilized Al3+ affinity chromatography and high performance liquid chromatography and subjected to amino acid sequence and mass spectrometric analyses. The molecular masses of the photolabeled products of cyanogen bromide cleavage were estimated using tricine-SDS-polyacrylamide gel electrophoresis. Overlapping sequence analysis localized the higher affinity site to the region corresponding to residues 289-291 and the other site to the region corresponding to residues 147-160. Site-directed mutagenesis of Cys290, a residue conserved in all known class III reductases, resulted in a protein that exhibited less than 10% of wild type enzymatic activity. These observations indicate that Cys290 may reside in or near the active site. High performance liquid chromatography analysis revealed that photoinsertion of [gamma-32P]8-N3ATP into the site corresponding to residues 147-160 was almost completely abolished when 100 microM dATP, dGTP, or dTTP was included in the photolabeling reaction mixture, whereas 100 microM ATP, GTP, CTP, or dCTP had virtually no effect. Based on these nucleotide binding properties, we conclude that this site is an allosteric site analogous to the one that has been shown to regulate substrate specificity of other ribonucleotide reductases. There was no evidence for a second allosteric nucleotide-binding site as observed in the anaerobic ribonucleotide reductase from Escherichia coli.
Highlights
May be striking differences in primary sequence, the same general mechanism for allosteric regulation appears to apply to all ribonucleotide reductases described to date, with only subtle differences observed [7]
Based on sequence homology (58% identity and 72% similarity) with the anaerobic reductase from E. coli [13] for which the allosteric properties have been described [11], it is postulated that the active site and the allosteric sites reside within the NrdD subunit
Kinetic evidence supports the existence of two different allosteric sites and an active site in the class III reductase from E. coli, the location of these nucleotide-binding sites has not been determined
Summary
Materials—[␥-32P]8-N3ATP1 was synthesized as described previously [20]. All other nucleotides were purchased from Amersham Pharmacia Biotech. Prior to photolabeling experiments, the DTT was removed by desalting on an Amersham Pharmacia Biotech NAP-5 column equilibrated with 50 mM NaPi, pH 7.0. All incubations and irradiations were carried out in microcentrifuge tubes over ice. After photolabeling, the protein was precipitated in 8% perchloric acid and washed with cold methanol. Saturation of T4 Anaerobic Ribonucleotide Reductase—1.5 g of NrdD was incubated with increasing concentrations of [␥-32P]8-N3ATP in the presence of 10 mM NaPi, pH 7.0, 5 mM MgCl2 and photolabeled as described above. Stoichiometry of photoinsertion of [␥-32P]8-N3ATP was determined using a NAP-5 column (Amersham Pharmacia Biotech) as described previously [22], except that the desalting was done in the presence of 10 mM dATP instead of ATP. The pellet was resolubilized in a solution of 6.0 M urea, 22 mM ammonium acetate, pH 8, and 10 mM 2-mercaptoethanol.
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