Kinetic scheme for intermolecular RNA cleavage by a ribozyme derived from hepatitis delta virus RNA.
A minimal kinetic mechanism for a trans-acting ribozyme derived from the HDV antigenomic RNA self-cleaving element was established from steady-state, pre-steady-state, single-turnover, and binding kinetics. Rate constants for individual steps, including substrate binding and dissociation, cleavage, and product release and binding, were measured at 37 degrees C at pH 8.0 in 10 mM Mg(2+) using oligonucleotides as either substrates, noncleavable analogues or 3' product mimics. A substrate containing a normal 3',5'-linkage was cleaved with a first-order rate constant (k(2)) of 0.91 min(-)(1). The association rate constant for the substrate to the ribozyme (2.1 x 10(7) M(-)(1) min(-)(1)) was at the lower range of the expected value for RNA duplex formation, and the substrate dissociated with a rate constant (1.4 min(-)(1)) slightly faster than that for cleavage. Thus the binary complex was not at equilibrium with free enzyme and substrate prior to the cleavage step. Following cleavage, product release was kinetically ordered in that the 5' product was released rapidly (>12 min(-)(1)) relative to the 3' product (6.0 x 10(-)(3) min(-)(1)). Rapid 5' product release and lack of a demonstrable binding site for the 5' product could contribute to the difficulty in establishing the ribozyme-catalyzed reverse reaction (ligation). Slow release of the 3' product was consistent with the extremely low turnover under steady-state conditions as 3' product dissociation was rate-limiting. The equilibrium dissociation constant for the substrate was 24-fold higher than that of the 3' cleavage product. A substrate with a 2',5'-linkage at the cleavage site was cleaved with a rate constant (k(2)) of 1.1 x 10(-)(2) min(-)(1). Thus, whereas cleavage of a 3',5'-linkage followed a Briggs-Haldane mechanism, 2', 5' cleavage followed a Michaelis-Menten mechanism.
Shih
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Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27705, USA.
Been
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D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D002384:Catalysis; D003698:Hepatitis Delta Virus / Q000201:enzymology* / Q000235:genetics; D006868:Hydrolysis; D006878:Hydroxides / Q000737:chemistry; D007700:Kinetics; D008956:Models, Chemical; D008969:Molecular Sequence Data; D016337:RNA, Catalytic / Q000737:chemistry* / Q000235:genetics; D012367:RNA, Viral / Q000737:chemistry* / Q000235:genetics; D013379:Substrate Specificity / Q000235:genetics
D006878:Hydroxides; D016337:RNA, Catalytic; D012367:RNA, Viral; C031356:hydroxide ion