Existence of efficient divalent metal ion-catalyzed and inefficient divalent metal ion-independent channels in reactions catalyzed by a hammerhead ribozyme.
The hammerhead ribozyme is generally accepted as a well characterized metalloenzyme. However, the precise nature of the interactions of the RNA with metal ions remains to be fully defined. Examination of metal ion-catalyzed hammerhead reactions at limited concentrations of metal ions is useful for evaluation of the role of metal ions, as demonstrated in this study. At concentrations of Mn2+ ions from 0.3 to 3 mM, addition of the ribozyme to the reaction mixture under single-turnover conditions enhances the reaction with the product reaching a fixed maximum level. Further addition of the ribozyme inhibits the reaction, demonstrating that a certain number of divalent metal ions is required for proper folding and also for catalysis. At extremely high concentrations, monovalent ions, such as Na+ ions, can also serve as cofactors in hammerhead ribozyme-catalyzed reactions. However, the catalytic efficiency of monovalent ions is extremely low and, thus, high concentrations are required. Furthermore, addition of monovalent ions to divalent metal ion-catalyzed hammerhead reactions inhibits the divalent metal ion-catalyzed reactions, suggesting that the more desirable divalent metal ion-ribozyme complexes are converted to less desirable monovalent metal ion-ribozyme complexes via removal of divalent metal ions, which serve as a structural support in the ribozyme complex. Even though two channels appear to exist, namely an efficient divalent metal ion-catalyzed channel and an inefficient monovalent metal ion-catalyzed channel, it is clear that, under physiological conditions, hammerhead ribozymes are metalloenzymes that act via the significantly more efficient divalent metal ion-dependent channel. Moreover, the observed kinetic data are consistent with Lilley's and DeRose's two-phase folding model that was based on ground state structure analyses.
Zhou
Jing-Min
JM
Gene Discovery Research Center, National Institute of Advanced Industrial Science and Technology (AIST), Central 4, 1-1-1 Higashi, Tsukuba Science City 305-8562, Japan.
Zhou
De-Min
DM
Takagi
Yasuomi
Y
Kasai
Yasuhiro
Y
Inoue
Atsushi
A
Baba
Tadashi
T
Taira
Kazunari
K
D001483:Base Sequence; D002384:Catalysis / Q000187:drug effects; D002413:Cations, Divalent / Q000378:metabolism* / Q000494:pharmacology; D002414:Cations, Monovalent / Q000378:metabolism* / Q000494:pharmacology; D004578:Electron Spin Resonance Spectroscopy; D004735:Energy Transfer; D005453:Fluorescence; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism / Q000494:pharmacology; D008345:Manganese / Q000378:metabolism / Q000494:pharmacology; D008670:Metals / Q000378:metabolism* / Q000494:pharmacology; D009690:Nucleic Acid Conformation; D011522:Protons; D016337:RNA, Catalytic / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012964:Sodium / Q000378:metabolism / Q000494:pharmacology; D017186:Titrimetry
D002413:Cations, Divalent; D002414:Cations, Monovalent; D008670:Metals; D011522:Protons; D016337:RNA, Catalytic; C427204:hammerhead ribozyme; D008345:Manganese; D012964:Sodium; D008274:Magnesium