Pb. Conibear et al., KINETIC AND SPECTROSCOPIC CHARACTERIZATION OF FLUORESCENT RIBOSE-MODIFIED ATP ANALOGS UPON INTERACTION WITH SKELETAL-MUSCLE MYOSIN SUBFRAGMENT-1, Biochemistry, 35(7), 1996, pp. 2299-2308
The interaction of the fluorescent ATP analog 2-[3-(5-fluoresceinyl)th
ioureido]-ethyl]carbamoyl] adenosine 5'-triphosphate (FEDA-ATP) with r
abbit skeletal myosin subfragment 1 (S1) and acto-S1 was studied. This
and related ATP analogs are potentially useful for determination of t
he ATPase activity of single myosin filaments using fluorescence micro
scopy [Sowerby et al. (1993) J. Mel. Biol. 234, 114-123]. However, it
is neccesary that such analogs mimic ATP in their kinetics of turnover
. The apparent second-order association rate constants for FEDA-ATP bi
nding to S1 and for FEDA-ATP-induced dissociation of acto-S1 are about
4 times slower than those for ATP. As with ATP, the hydrolysis step i
s fast, so that the M . FEDA-ADP . P-i complex is the major steady-sta
te intermediate. The turnover rate is the same for the 2' and 3' FEDA-
ATP derivatives and similar to that of ATP itself. The dissociation ra
te constant for FEDA-ADP from S1 is identical to that for ADP. Actin-a
ctivated turnover is comparable for both FEDA-ATP and ATP. The corresp
onding rhodamine and sulfoindocyanine, Cy3.18 (Cy3), derivatives also
appear to be reasonable analogs. FEDA-ATP binding leads to a 25-40% re
duction in fluorescein fluorescence. Spectral properties of the bound
nucleotide were explored by trapping FEDA-ADP as its aluminum fluoride
complex. The fluorescence quenching is a consequence of a reduction i
n both absorbance and excited-state lifetime, but there is little chan
ge in spectral shape.