Transient kinetics of the interaction of 1,N6-ethenoadenosine 5'-triphosphate with myosin subfragment 1 under normal and cryoenzymic conditions: a comparison with adenosine 5'-triphosphate.
The kinetics of the interaction of the fluorescent analogue 1,N6-ethenoadenosine 5'-triphosphate (epsilon-ATP) with myosin subfragment 1 (S1) were studied at 15 and -7.5 degrees C with 40% ethylene glycol as cryosolvent. Two techniques were used: fluorescence stopped flow and rapid flow-quench. When S1 is mixed with epsilon-ATP in a stopped-flow apparatus, biphasic fluorescence transients are obtained which are difficult to assign. Chemical sampling by the rapid-flow-quench method led to the chemical identity and the kinetics of interconversion of key intermediates, and by this method the optical signals were assigned and information about the cleavage and release of products was obtained. The data were interpreted by a shortened form of the Bagshaw-Trentham scheme for myosin adenosinetriphosphatase: M + ATP K1 in equilibrium M.ATP k2----M*.ATP k3 in equilibrium k3 M**.ADP.Pi k4----M + ADP + Pi The constants obtained were compared with those for ATP under identical conditions. In agreement with Rosenfeld and Taylor [Rosenfeld, S. S., & Taylor, E. W. (1984) J. Biol. Chem. 259, 11920-11929] we find that epsilon-ATP is bound tightly to S1 and that the chemical step is slower than with ATP. We show that the fast fluorescence transient is due to the tight binding of epsilon-ATP with K1 = 32 microM and k2 = 58 s-1 at 15 degrees C. With ATP these values are 8 microM and 16 s-1, respectively. There is a large difference in the delta H for k2: 50 kJ.mol-1 for epsilon-ATP and 119 kJ.mol-1 for ATP.(ABSTRACT TRUNCATED AT 250 WORDS)
Tesi
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INSERM U 128, CNRS, Montpellier, France.
Travers
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Barman
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D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism*; D004985:Ethenoadenosine Triphosphate / Q000378:metabolism*; D007700:Kinetics; D008433:Mathematics; D008962:Models, Theoretical; D015879:Myosin Subfragments; D009218:Myosins / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism*; D011485:Protein Binding; D013050:Spectrometry, Fluorescence
D004985:Ethenoadenosine Triphosphate; D015879:Myosin Subfragments; D010446:Peptide Fragments; D000255:Adenosine Triphosphate; D009218:Myosins