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 RNase RNAP Rh, a derivative of RNase Rh, and its mutant enzymes, i.e., RNase RNAP Rh H104F, RNase RNAP Rh E105Q, and RNase RNAP Rh D51N. Based on comparisons of their profiles we concluded that protonation of His104 is indispensable for the enzymatic activity and Glu105 accelerates the 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 mechanisms of RNase Rh action. (i) His109 participates in enzymatic action as a general base catalyst which removes the hydrogen of the 2'-OH of the ribose moiety. (ii) His46 participates in the reaction 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) His104 interacts with phosphate anion and its protonation is favorable for the enzymatic activity. (iv) Since the protonated form of Glu105 is more favorable for activity, we postulate two possible roles for Glu105: (a) its stabilizes the intermediate, and (b) it interacts with the oxygen atom of P = O and polarizes the phosphorus atom.
Irie
M
M
Department of Microbiology, Hoshi College of Pharmacy, Tokyo.
Ohgi
K
K
Watanabe
H
H
Iwama
M
M
Nakamura
K T
KT
Kurihara
H
H
Nonaka
T
T
Mitsui
Y
Y
Horiuchi
H
H
Takagi
M
M
D015226:Dinucleoside Phosphates / Q000378:metabolism*; D004722:Endoribonucleases / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009154:Mutation; D012233:Rhizopus / Q000201:enzymology* / Q000235:genetics
D015226:Dinucleoside Phosphates; C026519:uridine monophosphate-uridine; D004722:Endoribonucleases; C075842:ribonuclease Rh