title: Escherichia coli ribonucleotide reductase. Radical susceptibility to hydroxyurea is dependent on the regulatory state of the enzyme.
abstract: Ribonucleotide reductase catalyzes the reduction of ribonucleotides to their corresponding deoxyribonucleotides via a radical-mediated mechanism. The enzyme from Escherichia coli consists of the two non-identical proteins, R1 and R2, the latter of which contains the necessary free radical located to a tyrosine residue. The radical scavenger hydroxyurea was found to reduce the tyrosyl radical of R2 in a second-order reaction. The rate constant (0.50 M-1 s-1 at 25 degrees C) for this process was several orders of magnitude lower than the hydroxyurea-dependent reduction of free tyrosyl radicals in solution. This difference probably reflects the fact that the R2 tyrosyl radical is buried in the interior of the protein. Formation of the R1R2 complex changed the susceptibility of the radical to hydroxyurea in a manner that reflects the regulatory state of the holoenzyme. Furthermore, binding of substrate or product to the holoenzyme complex made the R2 radical at least 10 times more susceptible to inactivation by hydroxyurea than it was in the isolated R2 protein. One active site mutation in the R1 protein was shown to affect the sensitivity of the tyrosyl radical of R2 differently than wild type protein R1 does. Our results clearly show that the susceptibility of the tyrosyl radical in R2 to inactivation by hydroxyurea can be used as an efficient probe for the regulatory state of the holoenzyme complex.
authors.affiliation: Department of Molecular Biology, Stockholm University, Sweden.
authors.forename: M
authors.initials: M
authors.lastname: Karlsson
authors.forename: M
authors.initials: M
authors.lastname: Sahlin
authors.forename: B M
authors.initials: BM
authors.lastname: Sjöberg
mesh_terms: D004926:Escherichia coli / Q000201:enzymology*; D006918:Hydroxyurea / Q000494:pharmacology*; D007700:Kinetics; D009154:Mutation; D012264:Ribonucleotide Reductases / Q000037:antagonists & inhibitors* / Q000235:genetics; D013696:Temperature
chemical_list: D012264:Ribonucleotide Reductases; D006918:Hydroxyurea