Copper•Lys-Gly-His-Lys mediated cleavage of tRNAPhe: Studies of reaction mechanism and cleavage specificity

The reactivity of [Cu2+center dot Lys-Gly-His-Lys-NH2](2+) and [Cu2+center dot Lys-Gly-His-Lys](+) toward tRNA(Phe) has been evaluated. The amidated and carboxylate forms of the copper peptides display complex binding behavior with strong and weak sites evident (K-D1(app) similar to 71 mu M, K-D2(app) similar to 211 mu M for the amide form; and K-D1(app) similar to 34 mu M, K-D2(app) similar to 540 mu M for the carboxylate form), while Cu2+(aq) yielded K-D1(app) similar to 81 mu M and K-D2(app) similar to similar to 136 mu M. The time-dependence of the reaction of [Cu2+center dot Lys-Gly-His-Lys](+) and [Cu2+center dot Lys-Gly- His-Lys-NH2](2+) with tRNA(Phe) yielded k(obs) similar to 0.075 h(-1) for both complexes. HPLC analysis of the reaction products demonstrated guanine as the sole base product. Mass spectrometric data shows a limited number of cleavage fragments with product peak masses consistent with chemistry occurring at a discrete site defined by the structurally contiguous D and T Psi C loops, and in a domain where high affinity magnesium centers have previously been observed to promote hydrolysis of the tRNA(Phe) backbone. This cleavage pattern is more selective than that previously observed by Long and coworkers for nickel complexes of a series of C-terminally amidated peptides (Gly-Gly-His, Lys-Gly-His, and Arg-Gly-His), and may reflect variations in structural recognition and a distinct reaction path by the nickel derivatives. The data emphasizes the optimal positioning of the metal-associated reactive oxygen species, relative to scissile bonds, as a major criterion for development of efficient catalytic nucleases or therapeutics. (C) 2009 Published by Elsevier Inc.