The MgADP-induced decrease of the SH1-SH2 fluorescence resonance energy transfer distance of myosin subfragment 1 occurs in two kinetic steps.
The fluorescence resonance energy transfer distance between 5-[2-[iodoacetyl)amino)ethyl]aminoaphthalene-1-sulfonic acid covalently attached to the SH1 thiol of myosin subfragment 1 as the energy donor and N-(4-dimethylamino-3,5-dinitrophenyl)maleimide attached to SH2 as the energy acceptor has been found to decrease by about 7 A in the presence of MgADP (Dalby, R. E., Weiel, J., and Yount, R. G. (1983) Biochemistry 22, 4696-4706; Cheung, H. C., Gonsoulin, F., and Garland, F. (1985) Biochim. Biophys. Acta 832, 52-62). Fluorescence stopped-flow experiments on the same system have yielded biphasic traces which are resolvable into a fast and slow component, k1 and k2, respectively. Results of experiments in which k1 and k2 were measured as a function of excess ADP concentration showed: 1) a nonlinear dependence of the apparent rate constants on [ADP]; 2) k1 is a factor of 10 faster than k2. These results are consistent with the 3-step mechanism previously proposed for nucleotide binding to myosin S1 (Garland, F., and Cheung, H. C. (1979) Biochemistry 18, 5281-5289). Kinetic experiments in which the anisotropy of the donor was monitored show this quantity to be unchanged over the course of the reaction. The biphasic decrease of donor intensity is assigned to an increase in energy transfer efficiency which, from the above results, is due to a decrease in donor-acceptor distance, occurring in two steps. The fast step is associated with a 4-5-A decrease of the donor-acceptor separation, while the slow step is associated with a further decrease of approximately 2 A.
Garland
F
F
Department of Natural Sciences, University of Michigan-Dearborn 48128.
Gonsoulin
F
F
Cheung
H C
HC
D000244:Adenosine Diphosphate / Q000494:pharmacology*; D000818:Animals; D004735:Energy Transfer; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology*; D008301:Maleimides; D015879:Myosin Subfragments; D009218:Myosins; D009282:Naphthalenesulfonates; D010446:Peptide Fragments; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D013438:Sulfhydryl Compounds
D008301:Maleimides; D015879:Myosin Subfragments; D009282:Naphthalenesulfonates; D010446:Peptide Fragments; D013438:Sulfhydryl Compounds; C016399:N-(4-dimethylamino-3,5-dinitrophenyl)maleimide; D000244:Adenosine Diphosphate; C006421:1,5-I-AEDANS; D009218:Myosins; D008274:Magnesium