Cryoenzymology of the hammerhead ribozyme.
The technique of cryoenzymology has been applied to the hammerhead ribozyme in an attempt to uncover a structural rearrangement step prior to cleavage. Several cryosolvents were tested and 40% (v/v) methanol in water was found to perturb the system only minimally. This solvent allowed the measurement of ribozyme activity between 30 and -33 degrees C. Eyring plots are linear down to -27 degrees C, but a drastic reduction in activity occurs below this temperature. However, even at extremely low temperatures, the rate is still quite pH dependent, suggesting that the chemical step rather than a structural rearrangement is still rate-limiting. The nonlinearity of the Eyring plot may be the result of a transition to a cold-denatured state or a glassed state.
Department of Chemistry and Biochemistry, University of Colorado, Boulder 80309, USA.
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Feig
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Ammons
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Uhlenbeck
D003080:Cold Temperature; D003451:Cryoprotective Agents; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D000432:Methanol; D009843:Oligoribonucleotides / Q000138:chemical synthesis; D016337:RNA, Catalytic / Q000737:chemistry*
D003451:Cryoprotective Agents; D009843:Oligoribonucleotides; D016337:RNA, Catalytic; D000432:Methanol