title: Kinetic studies on the reduction of the tyrosyl radical of the R2 subunit of E. coli ribonucleotide reductase.
abstract: Kinetic studies at 25 degrees C, I = 0.100 M (NaCl), on the reduction of the tyrosyl radical of the R2 protein of E. coli ribonucleotide reductase with hydroxyurea (HU), N-methylhydroxylamine, catechol, and seven hydroxamic acid derivatives are reported. There are no pH-dependences in the range 6.2-8.6 investigated except that introduced with N-methylhydroxylamine which itself protonates in this range. At pH 7.6 the rate constant (0.46 M-1 s-1) for the HU reaction is in agreement with earlier values. Slower reactions are observed with the bulkier acetohydroxamic (0.020 M-1 s-1) and benzohydroxamic acids (0.040 M-1 s-1). In the case of N-methylhydroxylamine the rate constant (0.41 M-1 s-1 at pH 7.6) decreases with pH, and it is concluded that the protonated form CH3NH2+OH(pKa = 6.2) has little or no reactivity with Tyr. For this reaction under air-free conditions a second-stage (0.027 M-1 s-1) corresponding to reduction of Fe(III)2 is observed. Mid-point redox potentials for the reductants and estimates of reduction potentials applying in the case of the protein are considered. The reactions with 1,2-dihydroxybenzene (catechol) and 3,4-dihydroxybenzohydroxamic acid (Didox) also have two stages, when the initial Tyr reduction, rate constants/M-1 s-1 for catechol (3.2) and Didox (0.010), is followed by removal of the Fe(III) to give catechol and catechol like Fe(III)-complexed products. The single stage reactions of the hydroxamic acid derivatives which incorporate charged amino-acid groups L-glutamic acid, L-histidine, L-glycine and L-lysine, are slow, and saturation kinetics are observed consistent with association (small K values) prior to redox. The mechanism of reduction of R2-Tyr by all of the reagents studied is discussed.
authors.affiliation: Department of Chemistry, University of Newcastle, Newcastle upon Tyne, UK.
authors.forename: J C
authors.initials: JC
authors.lastname: Swarts
authors.forename: M A
authors.initials: MA
authors.lastname: Aquino
authors.forename: J Y
authors.initials: JY
authors.lastname: Han
authors.forename: K Y
authors.initials: KY
authors.lastname: Lam
authors.forename: A G
authors.initials: AG
authors.lastname: Sykes
mesh_terms: D002021:Buffers; D002396:Catechols; D004926:Escherichia coli / Q000201:enzymology*; D005609:Free Radicals; D005971:Glutamates; D006863:Hydrogen-Ion Concentration; D006877:Hydroxamic Acids; D006898:Hydroxylamines; D006918:Hydroxyurea; D007700:Kinetics; D010084:Oxidation-Reduction; D012264:Ribonucleotide Reductases / Q000737:chemistry*; D013816:Thermodynamics; D014443:Tyrosine
chemical_list: D002021:Buffers; D002396:Catechols; D005609:Free Radicals; D005971:Glutamates; D006877:Hydroxamic Acids; D006898:Hydroxylamines; C050614:glutamate-gamma-hydroxamic acid; D014443:Tyrosine; D012264:Ribonucleotide Reductases; C034221:catechol; C018338:N-methylhydroxylamine; D006918:Hydroxyurea