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 niveus, 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 hydrolyzed these substrates at 0.7-4.5% of the rates of the native RNase Rh. The other mutants hydrolyzed 2',3'-cyclic nucleotides at 15-20% of the rates 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 1st step). In the presence of a 13-fold excess of uridine, H109F catalyzed the transphosphorylation reaction of 2',3'-cyclic AMP (A>p) to ApU. However, this reaction was not catalyzed by H46F mutant or native RNase Rh. These 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 by acting as a base catalyst to activate water and the 5'-hydroxyl group of nucleosides, respectively.
Irie
M
M
Department of Microbiology, Hoshi College of Pharmacy, Shinagawa-ku, Tokyo.
Ohgi
K
K
Iwama
M
M
Koizumi
M
M
Sasayama
E
E
Harada
K
K
Yano
Y
Y
Udagawa
J
J
Kawasaki
M
M
D001665:Binding Sites / Q000235:genetics / Q000502:physiology; D015226:Dinucleoside Phosphates / Q000096:biosynthesis; D004722:Endoribonucleases / Q000235:genetics / Q000378:metabolism*; D006639:Histidine / Q000235:genetics* / Q000502:physiology*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis / Q000187:drug effects; D007700:Kinetics; D009154:Mutation / Q000235:genetics / Q000502:physiology; D010766:Phosphorylation; D012233:Rhizopus / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics
D015226:Dinucleoside Phosphates; D006639:Histidine; D004722:Endoribonucleases; C075842:ribonuclease Rh