Abstract
1. The hydrolysis of 1, N6-ethenoadenosine triphosphate (εATP) by myosin [EC 3. 6. 1. 3] and the change in fluorescence of εATP induced by H-meromyosin were studied.
(a) The amount of initial burst (TCA-labile Pi formation) of εATP-splitting was 1.1-1.3 moles per mole (4.8×105g) of myosin. In the steady-state of the myosin-εATPase reaction, the maximum rate and the Michaelis-Menten constant were 0.13 sec-1 and 0.93μM, respectively.
(b) The excitation spectrum of the 410nm fluorescence of εATP was changed upon addition of H-meromyosin; the fluorescence intensity decreased in the 310nm region and increased in the 290nm region.
(c) Both the decrease and the increase in the fluorescence followed the same timecourse, including the recovery to the initial intensity. The rate of the change (either increase or decrease) in the εATP fluorescence was measured. The results obtained can be treated by using the Michaelis-Menten equation. The maximum rate and Michaelis-Menten constant were 7.4sec-1 and 36μM, respectively. The rate in the recovery phase was 0.1sec-1.
(d) The reaction steps and the assignment of the kinetic parameters obtained above are proposed to be as follows:
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Here, ES stands for a enzyme-substrate complex, and EεADPP for the myosin-phosphate-εADP complex, the formation of which accompanies the fluorescence change of εATP. The assignment of the parameters (0.13sec-1 and 0.93 μM) obtained for the phosphate liberation in the steady-state was unsettled at present.
2. The interaction of H-meromyosin and εATP was studied by measuring the difference absorption spectrum, the difference fluorescence emission spectrum and the iodide-quenching of fluorescence.
(a) The difference absorption spectrum of H-meromyosin induced by εATP was similar to that induced by ATP, but the former was about 80% of the latter in the magnitude.
(b) When excited at 288nm, the emission spectrum of the reaction mixture (εATP plus H-meromyosin) showed a decreased intensity in the 340nm region and an increased intensity in the 410nm region.
(c) Judging from the portion of fluorescence intensity at 340nm that was not quenched by KI, the amount of tryptophan residues of H-meromyosin “inaccessible” to iodide was increased by adding εATP. This increase is approximately 80% of the increase obtained with ATP.
(d) A quantitative comparison between the increase at 410nm and the decrease at 340nm indicated that the transfer of excitation energy occurs only from “accessible” (to iodide) tryptophan residues to the εATP bound to H-meromyosin.
