Kinetic Mechanism of MyosinV-S1 Using a New Fluorescent ATP Analogue ² Eva Forgacs, ‡ Suzanne Cartwright, ‡ Miha ´ly Kova ´cs, §,| Takeshi Sakamoto, § James R. Sellers, § John E. T. Corrie, ⊥ Martin R. Webb, ⊥ and Howard D. White* ,‡ Department of Physiological Sciences, Eastern Virginia Medical School, Norfolk, Virginia 23507, MRC National Institute for Medical Research, Mill Hill, London, NW7 1AA, United Kingdom, Laboratory of Molecular Physiology, National Heart, Lung and Blood Institute, Bethesda, Maryland 20892-1762, and Department of Biochemistry, Eo ¨tVo ¨s UniVersity, Pa ´ zma ´ ny stny.1/c, H-1117 Budapest, Hungary ReceiVed April 12, 2006; ReVised Manuscript ReceiVed August 11, 2006 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 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 (∼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. Actomyosin motors play a pivotal role in many cellular functions such as cellular transport, muscle contraction, and cell motility. The energy source of these cellular motor proteins is ATP hydrolysis. Actomyosin proceeds through a series of intermediates during the ATP hydrolysis cycle, which modulates the protein conformation and the interac- tion between actin and myosin. Several fluorescent ATP analogues such as ǫATP (1), azaATP (2), mantATP (3), deoxymant ATP (4), Cy3ATP, and Cy5ATP (5) have been used to aid in understanding the relationship between the ATP hydrolysis mechanism and the chemomechanical trans- duction. Nucleotides in which a fluorescent group is co- valently linked to the 3′- and/or 2′-position of the ribose have proven especially useful, as they have a hydrolysis mech- anism similar to ATP and a fluorescence emission that is sensitive to the altered environment produced upon binding to the active site of myosin (e.g., a 2-fold enhancement in emission with mant and deoxymantATP). A recent addition to the 3′-fluorescent ATP derivatives is deac-aminoATP, 1 in which the coumarin is coupled to 3′-amino-3′-deoxyATP by an amide linkage to produce a single, stable isomer. The increase in the fluorescence emission observed when deac- aminoATP or deac-aminoADP binds to skeletal myosinII is the largest (∼20-fold) of any of the nucleotide analogues tested to date. Measurements of the steady-state rate of deac-aminoATP hydrolysis and the rate of deac-aminoADP dissociation from rabbit skeletal actomyosin suggest that the hydrolysis mechanism is similar to that of the natural substrate, ATP (6). We have therefore made a detailed study of the mechanism of hydrolysis of deac-aminoATP by myosinV-S1 to determine if there are any significant changes ² This work was supported by EB00209 and a grant from the Carman Foundation. E.F. was supported by an AHA fellowship (0525531U). M.K. is supported by NIH Research Grant D43 TW006230 (1 R01 TW0072412S-1) funded by the Fogarty International Center and the National Heart, Lung and Blood Institute and an EMBO-HHMI Grant for Central Europe. T.S. was supported by the Japanese Society for the Promotion of Science Fellowship. J.E.T.C. and M.R.W. are supported by the MRC, UK. * Corresponding author. Phone: (757) 446-5652; fax: (757) 624- 2270; e-mail: whitehd@evms.edu. ‡ Eastern Virginia Medical School. § National Heart, Lung and Blood Institute. | Eo ¨tvo ¨s University. ⊥ MRC National Institute for Medical Research. 1 Abbreviations: actin, filamentous actin; myosinV-S1, myosinV subfragment 1; myosinV-HMM, heavy meromyosinV.; deac-amino- ATP, 3′-(7-diethylaminocoumarin-3-carbonylamino)-3′-deoxyadeno- sine-5′-triphosphate; ǫATP, 1,N 6 -ethenoadenosine-5′-triphosphate; aza- ATP, 1, N 6 -etheno-2-aza-ATP.; mantATP, 2′ (3′ )-O-(N-methylanthraniloyl)- adenosine-5′-triphosphate; deoxymantATP, 2′-deoxy-3′-mantATP; cy3ATP, 2′(3′)-O-[N-(2-(Cy3-amino)ethyl)carbamoyl]adenosine-5′- triphosphate; cy5ATP, 2′(3′)-O-[N-(2-(Cy5-amino)ethyl)carbamoyl]- adenosine 5′-triphosphate; MDCC-PBP, N-[2-(1-maleimidyl)ethyl]-7- diethylaminocoumarin-3-carboxamide labeled phosphate-binding protein. 13035 Biochemistry 2006, 45, 13035-13045 10.1021/bi060712n CCC: $33.50 © 2006 American Chemical Society Published on Web 10/10/2006