Multiple magnesium ions in the ribonuclease P reaction mechanism.
The ribozyme ribonuclease (RNase) P cleaves precursor transcripts to produce the mature 5'-end of tRNAs. This hydrolysis reaction has a divalent cation requirement that is primarily catalytic, rather than structural; RNase P can be considered a metalloenzyme. Kinetic analysis shows that the RNase P catalytic mechanism has a cooperative dependence upon Mg2+ concentration. At least three Mg2+ ions are required for optimal activity, suggesting a multiple metal ion mechanism. The 2'-OH at the site of substrate cleavage may act as a ligand for a catalytically important Mg2+: deoxyribose substitution reduces the apparent number of Mg2+ bound from three to two and increases the apparent dissociation constant for Mg2+ from the micromolar to the millimolar range. In addition to these cation effects, the deoxyribose substitution reduces the rate of catalysis by 3400-fold; substitution with 2'-O-methyl at the cleavage site reduces the catalytic rate 10(6)-fold. If we presume no significant conformational effects of the substitutions, these results suggest that the 2'-OH serves as hydrogen-bond donor. The kinetic analysis of the catalytic mechanism is based upon the characterization of the pH dependence of the reaction. There is a hyperbolic (saturable) dependence on hydroxide concentration, with the half-maximal rate achieved at pH 8.0-8.5. The rate of the cleavage step is about 200 min-1 at pH 8.0, which is 500-fold faster than the steady-state parameter kcat.
Department of Biology, Indiana University, Bloomington 47405.
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D002118:Calcium / Q000494:pharmacology; D002384:Catalysis; D002412:Cations; D003839:Deoxyadenosines / Q000378:metabolism; D004722:Endoribonucleases / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism* / Q000494:pharmacology; D016337:RNA, Catalytic / Q000378:metabolism*; D012360:RNA, Transfer, Phe / Q000378:metabolism; D043262:Ribonuclease P
D002412:Cations; D003839:Deoxyadenosines; D029968:Escherichia coli Proteins; D016337:RNA, Catalytic; D012360:RNA, Transfer, Phe; D004722:Endoribonucleases; D043262:Ribonuclease P; C470865:ribonuclease P, E coli; D008274:Magnesium; D002118:Calcium