Defining the catalytic metal ion interactions in the Tetrahymena ribozyme reaction.
Divalent metal ions play a crucial role in catalysis by many RNA and protein enzymes that carry out phosphoryl transfer reactions, and defining their interactions with substrates is critical for understanding the mechanism of biological phosphoryl transfer. Although a vast amount of structural work has identified metal ions bound at the active site of many phosphoryl transfer enzymes, the number of functional metal ions and the full complement of their catalytic interactions remain to be defined for any RNA or protein enzyme. Previously, thiophilic metal ion rescue and quantitative functional analyses identified the interactions of three active site metal ions with the 3'- and 2'-substrate atoms of the Tetrahymena group I ribozyme. We have now extended these approaches to probe the metal ion interactions with the nonbridging pro-S(P) oxygen of the reactive phosphoryl group. The results of this study combined with previous mechanistic work provide evidence for a novel assembly of catalytic interactions involving three active site metal ions. One metal ion coordinates the 3'-departing oxygen of the oligonucleotide substrate and the pro-S(P) oxygen of the reactive phosphoryl group; another metal ion coordinates the attacking 3'-oxygen of the guanosine nucleophile; a third metal ion bridges the 2'-hydroxyl of guanosine and the pro-S(P) oxygen of the reactive phosphoryl group. These results for the first time define a complete set of catalytic metal ion/substrate interactions for an RNA or protein enzyme catalyzing phosphoryl transfer.
Shan
S
S
Department of Biochemistry, B400 Beckman Center, Stanford University, Stanford, California 94305-5307, USA.
Kravchuk
A V
AV
Piccirilli
J A
JA
Herschlag
D
D
D000818:Animals; D001667:Binding, Competitive; D002104:Cadmium / Q000378:metabolism; D002384:Catalysis; D002413:Cations, Divalent / Q000378:metabolism; D008274:Magnesium / Q000378:metabolism; D008345:Manganese / Q000378:metabolism; D008670:Metals / Q000378:metabolism*; D010100:Oxygen / Q000378:metabolism; D016337:RNA, Catalytic / Q000378:metabolism*; D013379:Substrate Specificity; D013455:Sulfur / Q000378:metabolism; D013768:Tetrahymena / Q000201:enzymology*; D015032:Zinc / Q000378:metabolism
D002413:Cations, Divalent; D008670:Metals; D016337:RNA, Catalytic; D002104:Cadmium; D008345:Manganese; D013455:Sulfur; D008274:Magnesium; D015032:Zinc; D010100:Oxygen