Direct photoaffinity labeling of ribonucleotide reductase from Escherichia coli. Evidence for enhanced binding of the allosteric effector dTTP by the presence of substrates.

The B1 subunit of ribonucleotide reductase from Escherichia coli serves as the regulator for the production of deoxyribonucleotides, needed for DNA synthesis.The enzyme contains three types of nucleotide binding sites, the ribonucleoside diphosphate substrate sites and two classes of allosteric sites.The effectors dGTP and d m bind only to one class, while dATP and ATP bind to both classes.In this study, pure protein B1 was photoaffinity labeled with radioactive d l T P o r dATP, using the unmodified nucleotides and direct UV irradiation.The photoaffinity labeling with d l T P w a s 20-60-old more efficient than with d A T P , and maximally half of the allosteric sites on protein B1 could be labeled.The temperature optimum for photolabeling w a s 23 "C for dTTP and -70 "C for dATP.Competition experiments with an excess of nonradioactive nucleotide showed that the photoincorporation of dTTP or dATP could be inhibited only by the allosteric effector known t o bind to one class of regulatory sites on protein B1.One-dimensional peptide mapping indicated, however, that different peptides were labeled with d " P as compared to dATP.An unexpected stimulation (approximately %fold) of the photoincorporation of d r r P w a s found by the addition of the substrate GDP.This effect was temperature-dependent and occurred at a concentration of GDP (20-40 p ~) close to the ICdiss for this substrate.Addition of CDP or ADP stimulated the dlTP incorporation to a lower extent.Direct binding studies, using equilibrium dialysis, verified the positive effect of GDP on d'M" binding to protein B1 (changing the &iSs from 6 t o 2 p ~) .The possible role of this new type of effect for the regulation of ribonucleotide reductase is discussed.Ribonucleotide reductase is the enzyme responsible for reduction of ribonucleotides to the corresponding deoxyribonucleotides.The same enzyme has the critical role of maintaining an adequate and balanced supply of all the four DNA precursors.The well characterized ribonucleotide reductase from Escherichia coli is composed of two nonidentical subunits, proteins B1 and B2, both needed for activity.Protein B2 (M, = 78,000) contains nonheme iron and a tyrosine free radical essential for the enzymatic reaction (1, 2).