Characterization of the Active Site of Ribonucleotide Reductase of Escherichia coli , Bacteriophage T4 and Mammalian Cells by Inhibition Studies with Hydroxyurea Analogues

Hydroxyurea specifically inhibits the enzyme ribonucleotide reductase by inactivating the essential tyrosine free radical in one of the two non‐identical subunits constituting the enzyme. Studies on the reactivity of hydroxyurea analogues towards the free radical salt, potassium nitrosodisulphonate, and the radical‐containing B2 subunit of Escherichia coli ribonucleotide reductase showed that, to obtain maximal enzyme inhibition, the most important parameter of an analogue was the ability to undergo a one‐electron oxidation. Also, a common geometrical feature for the analogues with high inhibitory potency was the planarity of the molecules. Results of inhibition studies with analogues containing bulky substituents but having about the same ability to reduce the free radical salt suggested that the tyrosine free radical of protein B2 is located in a pocket about 0.4 nm wide and more than 0.6 nm deep The ribonucleotide reductases of E. coli , T‐4infected E. coli and mammalian cells all contain the same tyrosine free radical structure. Still the mammalian reductase showed a 75‐fold higher sensitivity to inhibition by 3,4‐dihydroxybenzohydroxamic acid than the E. coli reductase. In contrast, the sensitivity to hydroxyurea was the same for both enzymes. The dihydroxybenzohydroxamic acid and hydroxyurea both reacted immediately (t ½ > 5) with the free radical salt. Therefore the difference in sensitivity between the mammalian and bac‐ terial reductase most probably reflects different topologies of their active sites with the site of the mammalian enzyme being more exposed. The T4‐induced reductase showed a 10‐fold increased sensitivity towards both hydroxyurea and the poly‐ hydroxybenzohydroxamic acids compared to the E. coli enzyme, indicating a greater ability of the T4 tyrosine free radical to undergo a one‐electron reduction. A closer understanding of the different topologies of the active sites of different ribonucleotide reductases could be of great value in the design of target‐directed drugs.