Kinetic and spectroscopic characterization of fluorescent ribose-modified ATP analogs upon interaction with skeletal muscle myosin subfragment 1.
The interaction of fluorescent ATP analog 2'(3')-O-[N-[2-[3-(5-fluoresceinyl)thioureido]-ethyl]carbamoyl]adenosine 5'-triphosphate (FEDA-ATP) with rabbit skeletal myosin subfragment 1 (S1) and acto-S1 was studied. This and related ATP analogs are potentially useful for determination of the ATPase activity of single myosin filaments using fluorescence microscopy [Sowerby et al. (1993) J. Mol. Biol. 234, 114-123]. However, it is necessary that such analogs mimic ATP in their kinetics of turnover. The apparent second-order association rate constants for FEDA-ATP binding 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 is fast, so that the M.FEDA-ADP.P(i) complex is the major steady-state intermediate. The turnover rate is the same for the 2' and 3' FEDA-ATP derivatives and similar to that of ATP itself. The dissociation rate constant for FEDA-ADP from S1 is identical to that for ADP. Actin-activated turnover is comparable for both FEDA-ATP and ATP. The corresponding rhodamine and sulfoindocyanine, Cy3.18 (Cy3), derivatives also appear to be reasonable analogs. FEDA-ATP binding leads to a 25-40% reduction 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 in both absorbance and excited-state lifetime, but there is little change in spectral shape.
Conibear
P B
PB
Department of Biochemistry, University of Leicester, U.K.
Jeffreys
D S
DS
Seehra
C K
CK
Eaton
R J
RJ
Bagshaw
C R
CR
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D000818:Animals; D005452:Fluoresceins / Q000378:metabolism*; D005456:Fluorescent Dyes; D006868:Hydrolysis; D007536:Isomerism; D007700:Kinetics; D018482:Muscle, Skeletal / Q000378:metabolism*; D015879:Myosin Subfragments / Q000378:metabolism*; D011817:Rabbits; D012266:Ribose / Q000737:chemistry*; D013050:Spectrometry, Fluorescence
D005452:Fluoresceins; D005456:Fluorescent Dyes; D015879:Myosin Subfragments; C097960:caged FEDA-ATP; D012266:Ribose; D000255:Adenosine Triphosphate