Kinetic Mechanism of MyosinV-S1 Using a New Fluorescent ATP Analogue

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• 作者：Eva Forgacs ; Suzanne Cartwright ; Mih&aacute ; ly Kov&aacute ; cs ; Takeshi Sakamoto ; James R. Sellers ; John E. T. Corrie ; Martin R. Webb ; Howard D. White
• 刊名：Biochemistry
• 出版年：2006
• 出版时间：October 31, 2006
• 年：2006
• 卷：45
• 期：43
• 页码：13035 - 13045
• 全文大小：381K
• 年卷期：v.45,no.43(October 31, 2006)
• ISSN：1520-4995

文摘

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 singleheaded 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 largeincrease in fluorescence emission (~20-fold) upon binding to the myosinV active site. The fluorescenceemission intensity is independent of the hydrolysis state of the nucleotide bound to myosinV-S1. Thevery good signal-to-noise ratio that is obtained with deac-amino nucleotides makes them excellent substratesfor studying expressed proteins that can only be isolated in small quantities. The combination of the fastrate of binding and the favorable signal-to-noise ratio also allows deac-nucleotides to be used in chaseexperiments to determine the kinetics of ADP and Pi dissociation from actomyosin-ADP-Pi. Althoughphosphate dissociation from actomyosinV-ADP-Pi does not itself produce a fluorescence signal, it producesa lag in the signal for deac-aminoADP dissociation. The lag provides direct evidence that the principalpathway of product dissociation from actomyosinV-ADP-Pi is an ordered mechanism in which phosphateprecedes ADP. Although the mechanism of hydrolysis of deac-aminoATP by (acto)myosinV-S1 isqualitatively similar to the ATP hydrolysis mechanism, there are significant differences in some of therate constants. Deac-aminoATP binds 3-fold faster to myosinV-S1, and the rate of deac-aminoADPdissociation 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.