Kinetics of Radical Intermediate Formation and Deoxynucleotide Production in 3-Aminotyrosine-Substituted Escherlchia coli Ribonucleotide Reductases

摘要

Escherichia coli ribonucleotide reductase is an α2β2 complex and catalyzes the conversion of nudeoside 5'-dipho-sphates (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 (Y_(122)·) cofactor in β2. This oxidation occurs by a mechanism of long-range proton-coupled electron transfer (PCET) over 35 A through a specific pathway of residues: Y_(122)·→W_(48)→Y_(356) in β2 to Y_(731)→Y_(730)→ C_(439) in α2. To study the details of this process, 3-aminotyrosine (NH_2Y) has been site-specifically incorporated in place of Y_(356) of β.The resulting protein, Y_(356)NH_2Y-β2, and the previously generated proteins Y_(731)NH_2Y-α2 and Y_(730)NH_2Y-α2 (NH_2Y-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 NH_2Y-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, NH_2Y· formation is biphasic (k_fast of 9—46 s~(-1) and k_solw 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 NH_2Y· formation, while the k_cat of ~0.5 s~(-1) is proposed to be associated with rate-limiting oxidation by NH_2Y· of the subsequent amino acid on the pathway during forward PCET. The X-ray crystal structures of Y(730)NH_2Y-α2 and Y_(731)NH_2Y-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.