THE ANAEROBIC RIBONUCLEOTIDE REDUCTASE FROM ESCHERICHIA COLI, A ZINC PROTEIN. IMPORTANCE OF THE METAL-BINDING SITE FOR THE ENZYME STRUCTURE AND ACTIVATION.
LA RIBONUCLEOTIDE REDUCTASE ANAEROBIE D'ESCHERICHIA COLI, UNE PROTEINE A ZINC. IMPORTANCE DU SITE METALLIQUE POUR LA STRUCTURE ET L'ACTIVITE DE L'ENZYME.
Résumé
RIBONUCLEOTIDE REDUCTASES (RNRS) ARE CRITICAL ENZYMES FOR THE DE NOVO SYNTHESIS OF DNA IN ALL ORGANISMS. FOR ITS ANAEROBIC GROWTH, ESCHERICHIA COLI DEPENDS ON A CLASS III RNR, WHICH HARBORS IN ITS ACTIVE FORM AN OXYGEN-SENSITIVE FREE RADICAL LOCATED ON THE GLY681 RESIDUE, THE FORMATION OF WHICH INVOLVES THE CONCERTED ACTION OF FOUR COMPONENTS: AN ACTIVATING IRON-SULFUR PROTEIN, S-ADENOSYLMETHIONINE, DITHIOTHREITOL (DTT), AND A REDUCING SYSTEM. A ZN(CYS)4 CENTER HAS RECENTLY BEEN FOUND IN THE C-TERMINAL REGION OF THE CRYSTAL STRUCTURE OF THE RNR FROM BACTERIOPHAGE T4. IN THIS WORK WE DEFINE NEW CONDITIONS ALLOWING FOR THE PREPARATION OF A HIGH SPECIFIC ACTIVITY REDUCTASE. WE SHOW THAT THE ZINC SITE CONTROLS THE STRUCTURATION OF THE CTER LOOP CARRYING THE RADICAL SITE. FINALLY WE DEMONSTRATE THAT DTT IS NOT ESSENTIAL FOR RADICAL FORMATION BUT RATHER ACTS ON RADICAL TRANSFER REACTIONS BETWEEN GLY· AND THE SUBSTRATE.
LES RIBONUCLEOTIDES REDUCTASES (RNRS) SONT DES ENZYMES UBIQUITAIRES ESSENTIELLES POUR LA SYNTHESE D'ADN. LA CROISSANCE EN ANAEROBIOSE D'ESCHERICHIA COLI DEPEND D'UNE RNR DE CLASSE III. L'ENZYME ACTIVEE CONTIENT UN RADICAL SENSIBLE A L'OXYGENE SITUE SUR LE RESIDU G681 DONT LA FORMATION IMPLIQUE L'INTERVENTION CONCERTEE D'UNE PROTEINE ACTIVATRICE FER-SOUFRE, DE S-ADENOSYLMETHIONINE, DE DITHIOTHREITOL (DTT), ET D'UN SYSTEME REDUCTEUR. LA STRUCTURE CRISTALLOGRAPHIQUE DE LA RNR DU BACTERIOPHAGE T4 A REVELE LA PRESENCE D'UN SITE METALLIQUE ZN(CYS)4 DANS LA PARTIE C-TERMINALE DE LA REDUCTASE. DANS CE TRAVAIL NOUS AVONS DEFINI DE NOUVELLES CONDITIONS DE PURIFICATION CONDUISANT A DES ENZYMES TRES ACTIVES, MONTRE QUE LE ZINC CONTROLE LA STRUCTURATION DE LA BOUCLE CTER CONTENANT LE SITE RADICALAIRE ET QUE, CONTRAIREMENT A CE QUI ETAIT ADMIS DEPUIS PLUS DE 15 ANS, LE DTT N'INTERVIENT PAS DANS LA FORMATION DU RADICAL GLY· MAIS PLUTOT DANS LES TRANSFERTS RADICALAIRES ENTRE GLY· ET LE SUBSTRAT.
Domaines
Biochimie [q-bio.BM]
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