Kinetics of radical intermediate formation and deoxynucleotide production in 3-aminotyrosine-substituted Escherichia coli ribonucleotide reductases

Ellen C. Minnihan, Mohammad R. Seyedsayamdost, Ulla Uhlin, Joanne Stubbe

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43 Scopus citations

Abstract

Escherichia coli ribonucleotide reductase is an α2β2 complex and catalyzes the conversion of nucleoside 5′-diphosphates (NDPs) to 2′-deoxynucleotides (dNDPs). The reaction is initiated by the transient oxidation of an active-site cysteine (C439) in α2 by a stable diferric tyrosyl radical (Y122•) cofactor in β2. This oxidation occurs by a mechanism of long-range proton-coupled electron transfer (PCET) over 35 Å through a specific pathway of residues: Y 122•→ W48→ Y356 in β2 to Y731→ Y730→ C439 in α2. To study the details of this process, 3-aminotyrosine (NH2Y) has been site-specifically incorporated in place of Y356 of β. The resulting protein, Y356NH2Y-β2, and the previously generated proteins Y731NH2Y-α2 and Y 730NH2Y-α2 (NH2Y-RNRs) are shown to catalyze dNDP production in the presence of the second subunit, substrate (S), and allosteric effector (E) with turnover numbers of 0.2-0.7 s-1. Evidence acquired by three different methods indicates that the catalytic activity is inherent to NH2Y-RNRs and not the result of copurifying wt enzyme. The kinetics of formation of 3-aminotyrosyl radical (NH 2Y•) at position 356, 731, and 730 have been measured with all S/E pairs. In all cases, NH2Y• formation is biphasic (k fast of 9-46 s-1 and kslow of 1.5-5.0 s -1) and kinetically competent to be an intermediate in nucleotide reduction. The slow phase is proposed to report on the conformational gating of NH2Y• formation, while the kcat of ∼0.5 s -1 is proposed to be associated with rate-limiting oxidation by NH2Y• of the subsequent amino acid on the pathway during forward PCET. The X-ray crystal structures of Y730NH2Y-α2 and Y731NH2Y-α2 have been solved and indicate minimal structural changes relative to wt-α2. From the data, a kinetic model for PCET along the radical propagation pathway is proposed.

Original languageEnglish (US)
Pages (from-to)9430-9440
Number of pages11
JournalJournal of the American Chemical Society
Volume133
Issue number24
DOIs
StatePublished - Jun 22 2011

All Science Journal Classification (ASJC) codes

  • Catalysis
  • Chemistry(all)
  • Biochemistry
  • Colloid and Surface Chemistry

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