Kinetics of ADP dissociation from the trail and lead heads of actomyosin V following the power stroke.
Myosin V is a cellular motor protein, which transports cargos along actin filaments. It moves processively by 36-nm steps that require at least one of the two heads to be tightly bound to actin throughout the catalytic cycle. To elucidate the kinetic mechanism of processivity, we measured the rate of product release from the double-headed myosin V-HMM using a new ATP analogue, 3'-(7-diethylaminocoumarin-3-carbonylamino)-3'-deoxy-ATP (deac-aminoATP), which undergoes a 20-fold increase in fluorescence emission intensity when bound to the active site of myosin V (Forgacs, E., Cartwright, S., Kovács, M., Sakamoto, T., Sellers, J. R., Corrie, J. E. T., Webb, M. R., and White, H. D. (2006) Biochemistry 45, 13035-13045). The kinetics of ADP and deac-aminoADP dissociation from actomyosin V-HMM, following the power stroke, were determined using double-mixing stopped-flow fluorescence. These used either deac-aminoATP as the substrate with ADP or ATP chase or alternatively ATP as the substrate with either a deac-aminoADP or deac-aminoATP chase. Both sets of experiments show that the observed rate of ADP or deac-aminoADP dissociation from the trail head of actomyosin V-HMM is the same as from actomyosin V-S1. The dissociation of ADP from the lead head is decreased by up to 250-fold.
Forgacs
Eva
E
Department of Physiological Sciences, Eastern Virginia Medical School, Norfolk, Virginia 23507, USA.
Cartwright
Suzanne
S
Sakamoto
Takeshi
T
Sellers
James R
JR
Corrie
John E T
JE
Webb
Martin R
MR
White
Howard D
HD
D000199:Actins / Q000302:isolation & purification / Q000378:metabolism; D000205:Actomyosin / Q000378:metabolism*; D000244:Adenosine Diphosphate / Q000378:metabolism*; D000818:Animals; D006868:Hydrolysis; D007700:Kinetics; D051379:Mice; D018482:Muscle, Skeletal / Q000378:metabolism; D015879:Myosin Subfragments / Q000378:metabolism; D024701:Myosin Type V / Q000378:metabolism*; D011817:Rabbits; D013050:Spectrometry, Fluorescence
D000199:Actins; D015879:Myosin Subfragments; D000244:Adenosine Diphosphate; D000205:Actomyosin; D024701:Myosin Type V