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

Abstract

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.degree. 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. The constants obtained were compared with those for ATP under identical conditions. In agreement with Rosenfeld and Taylor [Rosenfeld, S.S., and 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 .mu.M and k2 = 58 s-1 at 15.degree. C. With ATP these values are 8 .mu.M and 16 s-1, respectively. There is a large difference in the .DELTA.H.dbldag. for k2: 50 kJ .cntdot. mol-1 for .epsilon.-ATP and 119 kJ .cntdot. mol-1 for ATP. We suggest that the slow transient is due to a second type of nucleotide site which does not hydrolyze. At 15.degree. C with .epsilon.-ATP, k3 + k-3 = 1 s-1 and k4 = 1.3 s-1; with ATP the values were 22 s-1 and 0.12 s-1, respectively. Thus, whereas with ATP k4 is rate limiting, with .epsilon.-ATP no single step is clearly rate limiting. The kinetic constants were also obtained at -7.5.degree. C.