Ribonucleoside Diphosphate Reductase from E. coli

Ribonucleotide reductase of E. coli consists of two non-identical subunits, which are purified together during the initial steps of the purification procedure however upon further purification the reductase separates into two subunits, proteins B1 and B2 (58). The overall yield of the two proteins is low, in particular that of protein Bl, which readily decomposes into smaller subunits. This decomposition can be counteracted by the addition of dithiols. An improved procedure for the purification of protein B1 using affinity chromatography on dATP-Sepharose has been described by Thelander (59). 

Separately, protein Bl (mol. weight 160,000) and protein B2 (mol. weight 78,000) are inactive and do not catalyze any known partial reaction. However, in the presence of TTP, magnesium ions and dithio-threitol proteins Bl and B2 form a 1 1 complex (mol. weight 245,000) which is catalytically active (58, 59, 62, 63). 

When protein B1 and B2 are mixed in the presence of magnesium ions and dithiothreitol active ribonucleotide reductase with an S2o,w of 9.7S is formed. Stimulatory effectors, such as ATP and TTP, do not effect complex formation. In contrast, in the presence of the negative effector, dATP, at concentrations which inhibit enzyme activity a larger complex is formed with an S20, w of 15.5S. Both complexes contain equimolar amounts of each subunit. A heavy complex is also formed in the presence of mixtures of other nucleoside triphosphates which inhibit enzyme activity. On the other hand the formation of this heavy inactive complex is prevented by ATP at concentrations which reverse the inhibition by dATP (63). More recent experiments (59) have shown that the interaction between proteins B1 and B2 in the presence of dATP is strongly influenced by the presence of sucrose, and indeed in the absence of sucrose subunits B1 and B2 with dATP form a complex with an S20, w of 22.1S. 

Ribonucleoside Diphosphate Reductase Induced in E. coli by Infection with Bacteriophage T4 

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