Kinetic mechanism of myosin V-Sl using a new fluorescent ATP analogue

Eva Forgacs, Suzanne Cartwright, Mihály Kovács, Takeshi Sakamoto, James R. Sellers, John E.T. Corrie, Martin R. Webb, Howard D. White

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24 Citations (Scopus)

Abstract

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 myosin V-Sl. 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 (∼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-Sl. 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-Sl 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-Sl, and the rate of deac-aminoADP dissociation from actomyosinV-Sl 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.

Original languageEnglish
Pages (from-to)13035-13045
Number of pages11
JournalBiochemistry
Volume45
Issue number43
DOIs
Publication statusPublished - Oct 31 2006

ASJC Scopus subject areas

  • Biochemistry

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    Forgacs, E., Cartwright, S., Kovács, M., Sakamoto, T., Sellers, J. R., Corrie, J. E. T., Webb, M. R., & White, H. D. (2006). Kinetic mechanism of myosin V-Sl using a new fluorescent ATP analogue. Biochemistry, 45(43), 13035-13045. https://doi.org/10.1021/bi060712n