PH PROFILE OF KINETIC CONSTANTS OF RNASE RH FROM RHIZOPUS-NIVEUS AND ITS MUTANT ENZYMES TOWARDS UPU, AND POSSIBLE MECHANISMS OF RNASE RH

In order to elucidate the mechanism of action of Rhizopus niveus RNase Rh, we investigated the pH profiles of the kinetic parameters of RNas e RNAP Rh, a derivative of RNase Rh, and its mutant enzymes, i.e., RNa se RNAP Rh H104F, RNase RNAP Rh E105Q, and RNase RNAP Rh D51N. Based o n comparisons of their profiles we concluded that protonation of His10 4 is indispensable for the enzymatic activity and Glu105 accelerates t he enzymatic activity, especially at acid pH centered at pH 3.5. Based on these data and the previous data on the chemical modification and enzymatic properties of other mutant enzymes, we propose the following as a possible mechanism of RNase Rh action. (i) His109 participates i n enzymatic action as a general base catalyst which removes the hydrog en of the 2'-OH of the ribose moiety. (ii) His46 participates in the r eaction as a general acid catalyst which interacts with the 5'-oxygen atom of the scissile phosphodiester bond and becomes a proton donor to the departing nucleoside or nucleotide. (iii) Hisl04 interacts with p hosphate anion and its protonation is favorable for the enzymatic acti vity. (iv) Since the protonated form of Glu105 is more favorable for a ctivity, we postulate two possible roles for Glu105: (a) it stabilizes the intermediate, and (b) it interacts with the oxygen atom of P=O an d polarizes the phosphorus atom.