USE OF BINDING-ENERGY BY AN RNA ENZYME FOR CATALYSIS BY POSITIONING AND SUBSTRATE DESTABILIZATION

A fundamental catalytic principle for protein enzymes is the use of bi nding interactions away from the site of chemical transformation for c atalysis, We have compared the binding and reactivity of a series of o ligonucleotide substrates and products of the Tetrahymena ribozyme, wh ich catalyzes a site-specific phosphodiester cleavage reaction: CCCUCU pA + G reversible arrow CCCUCU-OH + GpA. The results suggest that this RNA enzyme, like protein enzymes, can utilize binding interactions to achieve substantial catalysis via entropic fixation and substrate des tabilization, The stronger binding of the all-ribose oligonucleotide p roduct compared to an analog with a terminal 3' deoxyribose residue gi ves an effective concentration of 2200 M for the 3' hydroxyl group, a value approaching those obtained with protein enzymes and suggesting t he presence of a structurally well defined active site capable of prec ise positioning. The stabilization from tertiary binding interactions is 40-fold less for the oligonucleotide substrate than the oligonucleo tide product, despite the presence of the reactive phosphoryl group in the substrate, This destabilization is accounted for by a model in wh ich tertiary interactions away from the site of bond cleavage position the electron-deficient 3' bridging phosphoryl oxygen of the oligonucl eotide substrate next to an electropositive Mg ion. As the phosphodies ter bond breaks and this 3' oxygen atom develops a negative charge in the transition state, the weak interaction of the substrate with Mg2becomes strong. These strategies of ''substrate destabilization'' and ''transition state stabilization'' provide estimated rate enhancements of approximate to 280- and approximate to 60-fold, respectively, Anal ogous substrate destabilization by a metal ion or hydrogen bond donor may be used more generally by RNA and protein enzymes catalyzing react ions of phosphate esters.