The RNA-cleaving bipartite DNAzyme is a distinctive metalloenzyme.
Much interest has focused on the mechanisms of the five naturally occurring self-cleaving ribozymes, which, in spite of catalyzing the same reaction, adopt divergent strategies. These ribozymes, with the exception of the recently described glmS ribozyme, do not absolutely require divalent metal ions for their catalytic chemistries in vitro. A mechanistic investigation of an in vitro-selected, RNA-cleaving DNA enzyme, the bipartite, which catalyzes the same chemistry as the five natural self-cleaving ribozymes, found a mechanism of significant complexity. The DNAzyme showed a bell-shaped pH profile. A dissection of metal usage indicated the involvement of two catalytically relevant magnesium ions for optimal activity. The DNAzyme was able to utilize manganese(II) as well as magnesium; however, with manganese it appeared to function complexed to either one or two of those cations. Titration with hexaamminecobalt(III) chloride inhibited the activity of the bipartite; this suggests that it is a metalloenzyme that utilizes metal hydroxide as a general base for activation of its nucleophile. Overall, the bipartite DNAzyme appeared to be kinetically distinct not only from the self-cleaving ribozymes but also from other in vitro-selected, RNA-cleaving deoxyribozymes, such as the 8-17, 10-23, and 614.
Feldman
Anat R
AR
Department of Molecular Biology and Biochemistry 1 and Department of Chemistry 2 Simon Fraser University, Burnaby, British Columbia V5A 1S6, Canada.
Leung
Edward K Y
EK
Bennet
Andrew J
AJ
Sen
Dipankar
D
D002384:Catalysis; D003035:Cobalt / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D021881:DNA, Catalytic / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry / Q000378:metabolism; D008345:Manganese / Q000737:chemistry / Q000378:metabolism; D012313:RNA / Q000737:chemistry* / Q000378:metabolism; D000091243:Ribosomal Protein S9; D012269:Ribosomal Proteins / Q000737:chemistry / Q000378:metabolism; D012997:Solvents / Q000737:chemistry
D021881:DNA, Catalytic; D000091243:Ribosomal Protein S9; D012269:Ribosomal Proteins; D012997:Solvents; C030862:cobalt ammonium complex; D003035:Cobalt; D008345:Manganese; D012313:RNA; D008274:Magnesium