Kinetic mechanism of myosinV-S1 using a new fluorescent ATP analogue.

@article{citeulike:2621155, 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 ( 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.}, address = {Department of Physiological Sciences, Eastern Virginia Medical School, Norfolk, Virginia 23507, USA.}, author = {Forgacs, E. and Cartwright, S. and Kov\'{a}cs, M. and Sakamoto, T. and Sellers, J. R. and Corrie, J. E. and Webb, M. R. and White, H. D. }, citeulike-article-id = {2621155}, doi = {http://dx.doi.org/10.1021/bi060712n}, issn = {0006-2960}, journal = {Biochemistry}, keywords = {fluorescent-nucleotide, kinetics, myosin, myosin-v}, month = {October}, number = {43}, pages = {13035--13045}, posted-at = {2008-04-01 23:46:48}, priority = {0}, title = {Kinetic mechanism of myosinV-S1 using a new fluorescent ATP analogue.}, url = {http://dx.doi.org/10.1021/bi060712n}, volume = {45}, year = {2006} }

TY - JOUR ID - citeulike:2621155 L3 - citeulike-article-id:2621155 TI - Kinetic mechanism of myosinV-S1 using a new fluorescent ATP analogue. JF - Biochemistry VL - 45 IS - 43 SP - 13035 EP - 13045 AD - Department of Physiological Sciences, Eastern Virginia Medical School, Norfolk, Virginia 23507, USA. SN - 0006-2960 N2 - 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. KW - fluorescent-nucleotide KW - kinetics KW - myosin KW - myosin-v AU - Forgacs, E AU - Cartwright, S AU - Kovács, M AU - Sakamoto, T AU - Sellers, JR AU - Corrie, JE AU - Webb, MR AU - White, HD PY - 2006/10/31/ UR - http://dx.doi.org/10.1021/bi060712n ER -