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°C.
Eyring plots are linear down to −27°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.