Heavy Meromyosin Mg-ATPase: Presteady-State and Steady-State H ⁺ Release

Abstract

The presteady state and steady state release of protons during hydrolysis of ATP by myosin has been studied by using a proteolytic subfragment of myosin, heavy meromyosin, isolated from rabbit skeletal muscle. Under the conditions of 0.5 M KCI, 9 mM MgCI2, pH 8, and 250, the rate of the H+ initial burst is proportional to the concentration of ATP added, and shows no plateau even when the concentration of ATP added is increased up to 750 ,AM. The apparent second order rate constant is approximately 7 X 105 M-1 sec1. When the concentration of ATP added is equal to or less than the concentration of heavy meromyosin catalytic sites, the H+ initial burst is followed by a slow exponential release of H+ with a rate constant of 0.017 sec'. This rate constant is equal to the steady state rate constant calculated for each heavy meromyosin catalytic site, suggesting that both sites split ATP independently. The magnitude of the H+ released per ATP bound is always 0.4 in the initial burst, and 0.6 in the slow exponential phase, which equals to a total of 1 H+ released per ATP hydrolyzed. On the basis of these observations, it is concluded that the H+ initial burst may well represent a fast ionization of H+ due to a conformational change as a result of nucleotide binding.It is now generally accepted that when ATP is added to rabbit skeletal myosin, heavy meromyosin (HMM), or subfragment-1 S-1, in the presence of excess Mg++, an initial rapid release of phosphate is observed prior to the onset of the steady state rate of phosphate release (1-3). Several workers have suggested that this initial phosphate burst might be correlated with the formation of an intermediate involved in the mechanism of muscle contraction in vivo (4-7). The rate of the initial phosphate burst has generally been reported to plateau at a rate of approximately 100 sec , but there is disagreement on the magnitude of the burst, with data varying from I to 2 mole of Pi released per mole of myosin or HMM (3,8,9 Eisenberg and Moos (17). The HMM was dialyzed against 0.5 M KCl before use. The stopflow experiments were performed using a two-syringe Aminco-Morrow stopflow mixer equipped with a 1-cm light path flow cell thermostated at 250C. The exhaust of the mixing chamber was slightly enlarged to reduce the flow resistance, which reduced the dead time of the instrument to approxiimately 2.1 msec for a reacting system containing 0.6-0.7 mg/ml of HMIM solution operating at 60 psi sampling pressure. The photodetection system was designed and built by Dr. P. 13. Chock and Mr. Ml.Greifner here at the National Institutes of Health. A detailed description of it will be published elsewhere. The time course

Magnesium ion dependent adenosine triphosphatase activity of heavy meromyosin as a function of temperature between +20 and -15.degree.C

“…In the second case, it has been shown that a transient proton release associated with the isomerization of heavy meromyosin occurs in the first steps of the hydrolysis at 20 °C (Koretz & Taylor, 1975;Chock & Eisenberg, 1974). This proton burst (-0.6 mol of H+/mol of enzyme at pH 8) is also observed at 20 °C or even at -7.5 °C, as shown in the present work, following the binding of various ATP analogues or ADP (Marsh et al, 1977) to the enzyme.…”

Binding and hydrolysis of ATP by cardiac myosin subfragment 1: effect of solution parameters on transient kinetics

“…The largest proton release would be observed at lower pHs, where the residue is largely protonated prior to reaction. Proton release accompanying the transient fluorescence enhancement has been measured for skeletal SI (Finlayson & Taylor, 1969;Chock & Eisenberg, 1974;Korentz & Taylor, 1975;Marsh et al, 1977;Chock, 1981), and the extent of proton release does decrease with increasing pH (Marsh et al, 1977). However, whether proton release occurs upon isomerization or cleavage has been a point of controversy.…”

Pre-steady-state kinetic evidence for a cyclic interaction of myosin subfragment one with actin during the hydrolysis of adenosine 5'-triphosphate

“…Stopped-Flow Apparatus. The stopped-flow apparatus used here essentially consists of a mercury-xenon arc light source powered with a Kepco JQE power supply, a Bausch and Lomb grating monochromator equipped with a manual lens shutter and adjustable slits, a thermostated two-syringe Aminco-Morrow stopped-flow pneumatic drive system equipped with a modified mixer flow cell (Chock and Eisenberg, 1974) the temperature of which was directly read via a Teflon probe attached to the outer wall of the observation flow cell and registered on a Yellow Spring Instrument tele-thermometer, an EMI 9558 QBS-20 photomultiplier tube powered by a Keithley 245 high-voltage supply, a fast interacting signal amplifying system (Rhee and Chock, 1976), and a Tektronix WP-1200 digital processing system where waveform averagings and computations were carried out.…”