Kinetic mechanism of myosinV-S1 using a new fluorescent ATP analogue.
We have used a new fluorescent ATP analogue, 3'-(7-diethylaminocoumarin-3-carbonylamino)-3'-deoxyadenosine-5'-triphosphate (deac-aminoATP), to study the ATP hydrolysis mechanism of the single headed myosinV-S1. Our study demonstrates that deac-aminoATP is an excellent substrate for these studies. Although the deac-amino nucleotides have a low quantum yield in free solution, there is a very large increase in fluorescence emission ( approximately 20-fold) upon binding to the myosinV active site. The fluorescence emission intensity is independent of the hydrolysis state of the nucleotide bound to myosinV-S1. The very good signal-to-noise ratio that is obtained with deac-amino nucleotides makes them excellent substrates for studying expressed proteins that can only be isolated in small quantities. The combination of the fast rate of binding and the favorable signal-to-noise ratio also allows deac-nucleotides to be used in chase experiments to determine the kinetics of ADP and Pi dissociation from actomyosin-ADP-Pi. Although phosphate dissociation from actomyosinV-ADP-Pi does not itself produce a fluorescence signal, it produces a lag in the signal for deac-aminoADP dissociation. The lag provides direct evidence that the principal pathway of product dissociation from actomyosinV-ADP-Pi is an ordered mechanism in which phosphate precedes ADP. Although the mechanism of hydrolysis of deac-aminoATP by (acto)myosinV-S1 is qualitatively similar to the ATP hydrolysis mechanism, there are significant differences in some of the rate constants. Deac-aminoATP binds 3-fold faster to myosinV-S1, and the rate of deac-aminoADP dissociation from actomyosinV-S1 is 20-fold slower. Deac-aminoATP supports motility by myosinV-HMM on actin at a rate consistent with the slower rate of deac-aminoADP dissociation.
Forgacs
Eva
E
Department of Physiological Sciences, Eastern Virginia Medical School, Norfolk, Virginia 23507, USA.
Cartwright
Suzanne
S
Kovács
Mihály
M
Sakamoto
Takeshi
T
Sellers
James R
JR
Corrie
John E T
JE
Webb
Martin R
MR
White
Howard D
HD
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism*; D000818:Animals; D006868:Hydrolysis; D007700:Kinetics; D051379:Mice; D015879:Myosin Subfragments / Q000378:metabolism*; D009218:Myosins / Q000378:metabolism*; D010710:Phosphates / Q000378:metabolism; D013050:Spectrometry, Fluorescence / Q000379:methods
D015879:Myosin Subfragments; D010710:Phosphates; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D009218:Myosins