Acid-Base Mechanism or a Triester-like Mechanism?

A general acid-base catalytic mechanism for ribonuclease A and other ribonucleases was previously widely accepted. However, an alternative to this mechanism was recently reintroduced in which attack by the 2’ hydroxyl group is facilitated by protonation of a nonbridging phosphoryl oxygen atom, instead of the leaving group oxygen atom, to give a triester-like mechanism of catalysis. Literature values for rate effects upon substitution of nonbridging phosphoryl oxygen atoms by sulfur (i.e., “thio-effects”) for enzymatic and nonenzymatic reactions of RNA and for nonenzymatic reactions of other phosphate diesters and triesters are compared herein. The thio-effects observed in the RNase A-catalyzed reactions are consistent with predictions based on the classical general acidbase catalyzed mechanism but are inconsistent with predictions based on the triester-like mechanism. Thus. the results of this analysis support the classical general acid-base mechanism rather than the triester-like mechanism. In addition, the results suggest that short, strong hydrogen bonds do not contribute substantially to RNase A catalysis. ’ Most biochemistry textbooks cite a general acid-base pathway for ribonuclease action. In this “classical” mechanism, one group (f-Iisl2 in RNase A) acts as a general base to remove the proton from the nucleophilic 2’ hydroxyl group and another group @-Iisll9 in RNase A) acts as a general acid to protonate the leaving group 5’ hydroxide ion in the transition state (Figure IA). However, this mechanism has been called into question with the proposal of a “triester-like” pathway,’ and at least one textbook has incorporated this mechanism.2 Ln this alternative mechanism. a nonbridging phosphoryl oxygen atom is protonated to render the substrate tnester-like (Figure 1B). The recent data suggested to support this pathway in the nonenzymatic cleavage of RNA have been questioned3 and . @Abstnct published in Advance ACS Abstructs, November 15, 1994. (1) Btrslow, R.; Xu. R. fruc. Nuall. Acud. Sci. LISA. 1993, 90, 1201(2) Abcles. R. H.; Frdy, P. A.; Jtncks, W. P. Biochetnistry, Jones and (3) Menger, F. M. J. Org. Clicm. 1991,56,6251. Haim. A. J . Am. Chem. 1207. Bartlett; Boston, MA, 1992; pp 884. SOC. 1992. 114. 8383-53S8. additional results pre~ented .~ Regardless of the final resolution of this controversy, the triester-like mechanism should be considered as a possible catalytic route for the enzyme-catalyzed reaction. Protonation of one of the nonbridging phosphoryl oxygens, to render the transition state more like that for a wiestcr reaction, provides a potential catalytic mechanism because phosphate triesters are typically more reactive than the corresponding phosphate diesters (-1plw-fold)?” Thus, whether or not there is substantiated evidence for this mechanism in solution, it represents a chemically reasonable proposal and therefore cannot be summarily dismissed for the enzymatic reaction. Indeed, even if the classical mechanism is followed in solution, there would be no guarantee that this mechanism would hold for the enzyme-catalyzed reaction. Information from X-ray crystallographic structures has been (4) Bre~low. R.; XU. R. J. Anr. Ciron. SOC. 1993. 115. 10705107 13. (5 ) Khan, S. A.: Kirby, A. J. J. Chenr. Soc. B 1970, 11721 182. (6) Chmdler, A. J.; Hollfeldcr, F.; Kirby, A. J.; O’Carroll. F.; Suombcrg. R. J . Chem. SOC., Perkin Trans. 2 1993, 327-333. OOO~-TS63!9~~/~~16-11631$0~.~0/0