Cryoenzymic studies on myosin subfragment 1: perturbation of an enzyme reaction by temperature and solvent

Abstract

The effects of temperature and solvent on myosin subfragment 1 ATPase have been studied. Under all of the conditions used the data could be fitted to the Bagshaw - Trentham pathway: (formula; see text) 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 degrees 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 approximately k+2 at low and approximately k+3 + k-3 at high temperatures) and steady-state experiments (kcat approximately k+6 at low and approximately k0 at high temperatures). A third cause is illustrated by k0: an Arrhenius plot of k0 is nonlinear since there is a break in K3. These studies illustrate 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.(ABSTRACT TRUNCATED AT 250 WORDS)

Nucleotide binding by myosin in glycerol‐extracted rabbit skeletal muscle fibres at temperatures between −35°C and 10°C

“…The present observations, combined with those of Biosca et al [6] confirm this difference and give a possible reason for its occurrence. For S1 there is a break in the van t'Hoff plot at + 15 "C, so that lowering the temperature below 15 "C shifts the hydrolytic equilibrium sharply towards ATP [6], whereas in fibres this is not so; down to -20°C the equilibrium remains almost unaltered (Fig. 1 B).…”

“…As the temperature was lowered below -22°C the ATP fraction rose sharply; by -35°C it predominated. These results are similar in type to those found by Biosca et al [(1984) Biochemistry 23, 1947-19531 on isolated subfragment 1, but are displaced to a much lower temperature range. Thus in a muscle fibre only a low thermal energy is needed for myosin to hold its nucleotide in a constant balance between ATP and ADP.…”

“…Actin contact also shifts the equilibrium towards ATP at room temperature [3]. By the use of ethyleneglycol to extend the available temperature range Biosca et al [6] have divided the effect of temperature into two regions: above 15°C it has little effect, while below 15°C the equilibrium is very sensitive to temperature. Ethyleneglycol itself does not significantly change the equilibrium [6] and leaves the chemical kinetics of ATP at the enzymatic site (but not its binding or the release of its products) little altered [7, 81. In both myofibrils and glycerol-extracted muscle fibres which have been relaxed by MgATP at low CaZ+ nucleotide binds to the enzymatic site of myosin [9-111.…”

Effect of solvent, pressure and temperature on reaction rates of the multiheme hydroxylamine oxidoreductase

“…mol-' respectively. Causes of nonlinear Arrhenius plots have been discussed by several authors and this phenomenon has been completely re-examined as it occurs in the reaction of myosin subfragment 1 ATPase [25]. Causes of defined breaks in an Arrhenius curve may include : (a) a temperature-induced conformation or 'phase' change in the enzyme; (b) a purely kinetic phenomenon, i.e., the replacement of one rate-limiting step by another at a critical temperature; (c) modification of the solvent structure at a critical temperature.…”

Dimethyl sulphoxide enhances the effects of Pi in myofibrils and inhibits the activity of rabbit skeletal muscle contractile proteins

“…2,3-Butanedione monoxime and a number of ATP and P i analogues have been exploited extensively with this objective [3][4][5][6][7][8][9][10][11][12][13]. Lowering the temperature has also provided valuable information, since the kinetics of some of the steps are affected more than others [14,15]. Ethylene glycol, which is used as an anti-freeze in most studies carried out at subzero temperatures, also modifies catalysis and cross-bridge kinetics in its own right, without changing structure or mechanism in any fundamental way [15][16][17][18][19][20][21][22].…”