Mechanisms of catalysis by imidazole buffers of the hydrolysis and isomerisation of RNA models

We report second-order rate constants for catalysis by 0.1-0.7 M imidazole-imidazolium (Im-ImH(+)) buffers of the hydrolysis and isomerisation of the dinucleoside monophosphate uridyl(3'-->5 ")uridine, a comparative study of the same reactions of the chimeric oligonucleotide TTUTT, and systematic measurements of medium and ionic strength effects on the reactions. At the concentrations used in this work: (i) the hydrolysis of 3',5 "-UpU is catalysed both by imidazole and, less effectively, by its conjugate acid, in reactions which are of the first order in the general acid or base. (ii) Isomerisation to 2',5 "-UpU is catalysed only by ImH(+), as is the isomerisation of TTUTT: there is no evidence for 'negative catalysis'. (iii) The hydrolysis of TTUTT is also catalysed by both imidazole and by its conjugate acid, but in this case catalysis by ImH(+) is the more effective. At constant ionic strength we see no sign of the bell-shaped dependence on the degree of protonation of the buffer seen previously with poly-U. (iv) The emergence of the 'bell-shape' in the reaction of UpU is shown to depend on the ionic strength of the medium. We conclude that the hydrolysis of the internucleoside bond in these phosphodiesters involves two, parallel, reaction pathways: (a) a more or less concerted general base catalysed reaction; and (b) a two-step process, involving the rate determining general acid catalysed breakdown of the phosphorane monoanion intermediate 10. Near pH 7, in the presence of Im and ImH(+), the two pathways fun at comparable, but very slow rates. Ribonucleases logically combine the catalytic advantages of both pathways.