Studies on Escherichia coli RNase P RNA with Zn2+ as the catalytic cofactor.
We demonstrate, for the first time, catalysis by Escherichia coli ribonuclease P (RNase P) RNA with Zn2+ as the sole divalent metal ion cofactor in the presence of ammonium, but not sodium or potassium salts. Hill analysis suggests a role for two or more Zn2+ ions in catalysis. Whereas Zn2+ destabilizes substrate ground state binding to an extent that precludes reliable Kd determination, Co(NH3)6(3+) and Sr2+ in particular, both unable to support catalysis by themselves, promote high-substrate affinity. Zn2+ and Co(NH3)6(3+) substantially reduce the fraction of precursor tRNA molecules capable of binding to RNase P RNA. Stimulating and inhibitory effects of Sr2+ on the ribozyme reaction with Zn2+ as cofactor could be rationalized by a model involving two Sr2+ ions (or two classes of Sr2+ ions). Both ions improve substrate affinity in a cooperative manner, but one of the two inhibits substrate conversion in a non-competitive mode with respect to the substrate and the Zn2+. A single 2'-fluoro modification at nt -1 of the substrate substantially weakened the inhibitory effect of Sr2+. Our results demonstrate that the studies on RNase P RNA with metal cofactors other than Mg2+ entail complex effects on structural equilibria of ribozyme and substrate RNAs as well as E*S formation apart from the catalytic performance.
Cuzic
Simona
S
Philipps-Universität Marburg, Institut für Pharmazeutische Chemie Marbacher Weg 6, D-35037 Marburg, Germany.
Hartmann
Roland K
RK
D001483:Base Sequence; D002384:Catalysis; D002413:Cations, Divalent; D003035:Cobalt / Q000737:chemistry; D003067:Coenzymes / Q000737:chemistry*; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D012343:RNA, Transfer / Q000737:chemistry / Q000378:metabolism; D043262:Ribonuclease P / Q000737:chemistry / Q000378:metabolism*; D013324:Strontium / Q000737:chemistry / Q000494:pharmacology; D015032:Zinc / Q000737:chemistry*
D002413:Cations, Divalent; D003067:Coenzymes; D029968:Escherichia coli Proteins; C030862:cobalt ammonium complex; D003035:Cobalt; D012343:RNA, Transfer; D043262:Ribonuclease P; C470865:ribonuclease P, E coli; D015032:Zinc; D013324:Strontium