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 HD V 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 Mg2+ using oligonucleotides as either substrates, noncleavable analo gues or 3' product mimics. A substrate containing a normal 3',5'-linkage wa s cleaved with a first-order rate constant (k(2)) of 0.91 min(-1). The asso ciation rate constant for the substrate to the ribozyme (2.1 x 10(7) M-1 mi n(-1)) was at the lower range of the expected value for RNA duplex formatio n, and the substrate dissociated with a rate constant (1.4 min(-1)) slightl y faster than that for cleavage. Thus the binary complex was not at equilib rium with free enzyme and substrate prior to the cleavage step. Following c leavage, 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) mi n(-1)). Rapid 5' product release and lack of a demonstrable binding site fo r the 5' product could contribute to the difficulty in establishing the rib ozyme-catalyzed reverse reaction (ligation). Slow release of the 3' product was consistent with the extremely low turnover under steady-state conditio ns as 3' product dissociation was rate-limiting. The equilibrium dissociati on constant for the substrate was 24-fold higher than that of the 3' cleava ge product. A substrate with a 2',5'-linkage at the cleavage site was cleav ed with a rate constant (k(2)) of 1.1 x 10(-2) min(-1). Thus, whereas cleav age of a 3',5'-linkage followed a Briggs-Haldane mechanism, 2',5' cleavage followed a Michaelis-Menten mechanism.