Localization and characterization of two nucleotide-binding sites on the anaerobic ribonucleotide reductase from bacteriophage T4

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.