ISSN:
1432-1327
Keywords:
Key words Ribonucleotide Reductase (Mouse)
;
Binuclear Fe2II I/Tyr
;
Enzyme
;
R2 subunit
;
Freeradical Enzyme Reactivity
;
Oxidants: hydroxyurea
;
hydrazine
;
phenylhydrazine
;
hydroxylamine
Source:
Springer Online Journal Archives 1860-2000
Topics:
Biology
,
Chemistry and Pharmacology
Notes:
Abstract Four reductions of the R2 subunit of mouse ribonucleotide reductase have been studied and found to exhibit different behaviour from that of Escherichia coli R2. An important difference is that there is no stable met-R2 (Fe2 II I) form of mouse R2. With hydroxyurea, hydrazine and hydroxylamine uniphasic kinetics are observed for the combined reduction of radical Tyr ˙ and Fe2 II I components to tyrosine and Fe2 II respectively. The rate constants, determined at 370 nm (emphasising FeIII decay) and 417 nm (emphasising Tyr ˙ decay), differ by factors of 2–3, allowing some mechanistic features to be defined. The studies with hydrazine are particularly important. In the case of E. coli R2, a first phase corresponding to two-equivalent reduction of the met-R2 component has been observed [18]. It is likely that the four times slower second phase reaction of active E. coli R2 also corresponds to the Fe2 II I → Fe2 II change and is followed by fast intramolecular Fe2 II reduction of the higher potential Tyr ˙. The latter changes are believed to hold also for (active) mouse R2. The FeIIFeIII semi forms have been detected at low levels by EPR for mouse R2 (9%) and E. coli (∼5%) in previous studies. Further substrate reduction of FeIIFeIII occurs at a comparable rate to account for the transient behaviour of FeIIFeIII. For mouse R2 the combined FeIII decay processes (which we are unable to separate) give smaller uniphasic rate constants at 370 nm than at 417 nm. A fitted-base-line (FBL) treatment of absorbance changes at 417 nm targets more closely the Tyr ˙ decay as a means of monitoring the rate-determining step. The FBL method gives rate constants k (M–1 s–1) at 25 °C and pH 7.5 for hydroxyurea (1.46), hydrazine (0.163) and hydroxylamine (4.4). Surprisingly, phenylhydrazine, with a less strong reduction potential (0.25 V), gives a substantially faster reduction of the Tyr ˙ as the only redox step (rate constant 27 M–1 s–1). In this case a slower second phase at 370 nm is independent of reductant and corresponds to rate-controlling release of FeIII. Overall the results indicate a more reactive redox centre for mouse R2 and help develop further an understanding of factors affecting the reactivity of R2.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1007/s007750050134
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