ROLE OF HISTIDINE-46 IN THE HYDROLYSIS AND THE REVERSE TRANSPHOSPHORYLATION REACTION OF RNASE RH FROM RHIZOPUS-NIVEUS

In order to study the reaction mechanism of RNase Rh from Rhizopus niv eus, the rates of cleavage of four 2',3'-cyclic nucleotides by mutant enzymes of RNase Rh, H46F, H109F, E105Q, and K108L were measured, H46F is virtually inactive towards cyclic nucleotides, but H109F hydrolyze d these substrates at 0.7-4.5% of the rates of the native RNase Rh, Th e other mutants hydrolyzed 2',3'-cyclic nucleotides at 15-20% of the r ates of the native enzyme, Relative enzymatic activities towards four cyclic nucleotides of H109F in the hydrolysis reaction (2nd step) were much higher than in the transphosphorylation reaction (the Ist step), In the presence of a 13-fold excess of uridine, H109F catalyzed the t ransphosphorylation reaction of 2',3'-cyclic AMP (A>p) to ApU, However , this reaction was not catalyzed by H46F mutant or native RNase Rh, T hese results showed that His46 is crucial to the hydrolysis reaction, and to the reversed reaction of the transphosphorylation reaction. We suggest that His46 in RNase Rh plays a major role in these reactions b y acting as a base catalyst to activate water and the 5'-hydroxyl grou p of nucleosides, respectively.