Copper.Lys-Gly-His-Lys mediated cleavage of tRNA(Phe): studies of reaction mechanism and cleavage specificity.
The reactivity of [Cu2+.Lys-Gly-His-Lys-NH2]2+ and [Cu2+.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) approximately 71 microM, K(D2)(app) approximately 211 microM for the amide form; and K(D1)(app) approximately 34 microM, K(D2)(app) approximately 240 microM for the carboxylate form), while Cu2+(aq) yielded K(D1)(app) approximately 81 microM and K(D2)(app) approximately 136 microM. The time-dependence of the reaction of [Cu2+.Lys-Gly-His-Lys]+ and [Cu2+.Lys-Gly-His-Lys-NH2]2+ with tRNA(Phe) yielded k(obs) approximately 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 TPsiC 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.
Bradford
Seth
S
Evans Laboratory of Chemistry, The Ohio State University, 100 West 18th Avenue, Columbus, OH 43210, USA.
Kawarasaki
Yuta
Y
Cowan
J A
JA
D000595:Amino Acid Sequence; D001665:Binding Sites; D003300:Copper; D006801:Humans; D006868:Hydrolysis / Q000187:drug effects; D007700:Kinetics; D013058:Mass Spectrometry; D009842:Oligopeptides / Q000494:pharmacology*; D012360:RNA, Transfer, Phe / Q000187:drug effects* / Q000378:metabolism; D013379:Substrate Specificity
D009842:Oligopeptides; D012360:RNA, Transfer, Phe; C020905:glycyl-histidyl-lysine; D003300:Copper