CRYOENZYMIC STUDIES ON MYOSIN SUBFRAGMENT-1 - PERTURBATION OF AN ENZYME REACTION BY TEMPERATURE AND SOLVENT

The effects of temperature and solvent on [rabbit] myosin subfragment 1 ATPase were studied. Under all of the conditions used the data could be fitted to the Bagshaw-Trentham pathway: .**GRAPHIC**. Ethylene glycol (40%) was used as the cryosolvent; this makes K1 and k+2 measurable and allows for temperature studies over an extensive temperature range (+35 to -20.degree. C) and thus to reasonably accurate thermodynamic parameters. The following techniques were used: ATP chase (for K1 and k+2); Pi burst (k+2 or k+3 + k-3); single-turnover Pi burst [k0 = k+4K3/(1 + K3)] absorption stopped flow (k+2 or k+3 + k-3); steady state (k+6 or k0). Myosin provides examples of causes for nonlinear Arrhenius and van''t Hoff plots. A temperature-induced structural change is exemplified by a jump in an Arrhenius plot of k+2 and breaks in van''t Hoff plots of K1 and K3. A change in rate-limiting step is illustrated from stopped-flow experiments (kobsd .apprx. k+2 at low and .apprx. k+3 + k-3 at high temperatures) and steady-state experiments (kcat .apprx. k+6 at low and .apprx. k0 at high temperatures). A 3rd case is illustrated by k0: an Arrhenius plot of k0 is nonlinear since there is a break in K3. The use of temperature perturbation as a way of revealing reaction intermediates and of defining the conditions required for the isolation of a particular intermediate is illustrated. Thus, at -15.degree. C K3 and K+4 are small and a key intermediate, M*.cntdot.ATP, accumulates with a half-life of 12 min. Unlike the preceding steps, step 4 (k+4) is little affected by the solvent (ethylene glycol, KCl) or by the temperature-induced structural change of myosin, and its .**GRAPHIC**. is low. The modulations of this step may be restricted to a specific interaction of M**.cntdot.ADP.cntdot.Pi with actin.