The hydrolysis of Mg2+-adenosine 5'-triphosphate (ATP) by heavy meromyosin has been studied between +20 and -15 degrees C, especially in the low-temperature range, in a medium containing 30% (v/v) ethylene glycol by fluorometric, spectrophotometric, and potentiometric measurements. The time course of the fluorescence changes of the enzyme during the reaction depends markedly on the temperature in consequence of large differences between the activation energies of the various steps. The observed kinetics have been analyzed according to the simplified scheme of Bagshaw & Trentham [Bagshaw, C. R., & Trentham, D. R. (1974) Biochem. J. 141, 331-349]. The following results have been obtained. (1) The rate-limiting step of the reaction changes in this temperature range; at 20 degrees C M**.ADP.Pi is the predominant steady-state complex, and M*.ADP predominates at -15 degrees C, with a half-life of approximately 10 min. (2) As expected, on the basis that it is the dissociation of the M*.ADP complex which becomes rate limiting at low temperature, one observes, in the pre-steady-state below 0 degrees C, both a proton burst and a lag phase in ADP release. (3) At low temperature, the equilibrium M*.ATP in equilibrium M**.ADP.Pi is displaced to the left All the kinetic data obtained in this study are compatible with a simple pathway for the Mg2+-ATP hydrolysis by myosin and with sequential release of the reaction products.
The effect of guanidine hydrochloride on ATPase activity, gel filtration, turbidity, exposure of thiol groups, far-UV circular dichroism, and the fluorescence emission intensity of myosin subfragment 1 (S-1) was studied under equilibrium conditions. It was found that the denaturation process involves several intermediate states. The enzymatic activity of S-1 is at first lost at very low concentrations of GdnHCl (lower than 0.5 M). At a slightly higher GdnHCl concentration (about 0.5 M), the light chains dissociate and this dissociation is closely followed by the formation of aggregates between the naked heavy chains of S-1 molecules in the guanidine hydrochloride range of concentrations 0.5-1 M. At GdnHCl concentrations above 1 M, aggregates gradually disappear and S-1 loses its secondary and tertiary structures. These phenomena are partly reversible, and ATPase activity is only partially recovered under highly limited conditions. These results are discussed in relation to the nature of myosin subunit assembly. The head fragment of 20 kDa is thus suggested to be implicated in the binding of light chain to heavy chain and in the self-association of free heavy chains.
The number of active sites of soluble and filamentous myosin and of its subfragments, heavy meromyosin and subfragment-1, has been determined. The titration involves steady-state kinetic measurements at a high enzyme concentration and varying substrate concentrations (or vice versa), in the presence of a substrateregenerating system. Some practical and theoretical conditions for its execution are given, and, in particular, the effect of a possible heterogeneity of the active sites on the titration curves is analysed. Under the experimental conditions of the study, the number of active sites is close to that of myosin heads, and the heads seem to be functionally identical ; the catalytic constants k,,, and K, characterizing each active site are similar within some limits (1 -2 for the ratio of k,,, values; 1 -5 for that of K , values).The determination of the absolute concentration of enzymes requires the titration of their active sites [l]. Ths is usually possible by combined steady-and presteady-state kinetic measurements, if an enzyme-bound intermediate accumulates during the reaction of the enzyme with its substrate and if a reaction product is concomitantly released. In the case where no intermediate accumulates, this active-site titration is not applicable.An alternative method involving steady-state kinetic measurements may be used if the apparent affinity of the enzyme for its substrate is high. In this case, the rate of the reaction at high enzyme concentration is maximum when the substrate concentration becomes equal to that of the concentration of enzyme active sites. Although this possibility has been known for a long time [2], it seems to have been hardly used, to our knowledge, except for a recent application to the active-site titration of soluble myosin and of its subfragments [3-61. This last study was simplified particularly by the regeneration of the substrate during the course of experiments due to an efficient substrate-regenerating system. In the present paper, we develop some theoretical and practical conditions for this titration as well as some pitfalls which may arise when it is carried out.Muscle myosins are double molecules made up of one pair of heavy chains and two pairs of light chains, the so-called alkali and regulatory subunits. In the case of fast-twitch muscle myosins, there are two different alkali subunits, the A1 and A2 chains, which have however no distinct effect on of two different heavy chains in an approximate ratio of 60:40. At the present time, the number and the identity of active sites of the two-headed myosin are still under debate [3,9 -121 and the question of the functional homogeneity or heterogeneity of these sites is not yet resolved. These points will be discussed from the results of active-site titrations of myosin and of its subfragments. THEORYSeveral authors have discussed steady-state enzyme kinetics with high concentrations of substrate and enzyme [2, 13 -161. For the following general enzyme reaction mechanism : E + S e E S .+ E + P , the initial rate ...
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