Mechanism of action of myosin X, a membrane-associated molecular motor.
We have performed a detailed biochemical kinetic and spectroscopic study on a recombinant myosin X head construct to establish a quantitative model of the enzymatic mechanism of this membrane-bound myosin. Our model shows that during steady-state ATP hydrolysis, myosin X exhibits a duty ratio (i.e. the fraction of the cycle time spent strongly bound to actin) of around 16%, but most of the remaining myosin heads are also actin-attached even at moderate actin concentrations in the so-called "weak" actin-binding states. Contrary to the high duty ratio motors myosin V and VI, the ADP release rate constant from actomyosin X is around five times greater than the maximal steady-state ATPase activity, and the kinetic partitioning between different weak actin-binding states is a major contributor to the rate limitation of the enzymatic cycle. Two different ADP states of myosin X are populated in the absence of actin, one of which shows very similar kinetic properties to actomyosin.ADP. The nucleotide-free complex of myosin X with actin shows unique spectral and biochemical characteristics, indicating a special mode of actomyosin interaction.
Kovács
Mihály
M
Laboratory of Molecular Physiology, NHLBI, National Institutes of Health, Bethesda, Maryland 20892-1762, USA. kovacsm@mail.nih.gov <kovacsm@mail.nih.gov>
Wang
Fei
F
Sellers
James R
JR
D000199:Actins / Q000737:chemistry; D000205:Actomyosin / Q000737:chemistry; D000244:Adenosine Diphosphate / Q000737:chemistry; D000251:Adenosine Triphosphatases / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D006868:Hydrolysis; D007313:Insecta; D007700:Kinetics; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D018995:Myosin Heavy Chains / Q000737:chemistry; D024701:Myosin Type V / Q000737:chemistry; D009218:Myosins / Q000737:chemistry* / Q000378:metabolism; D009711:Nucleotides / Q000737:chemistry; D010710:Phosphates / Q000737:chemistry; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D013997:Time Factors
D000199:Actins; D018076:DNA, Complementary; D009711:Nucleotides; D010710:Phosphates; C089785:myosin VI; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000205:Actomyosin; D000251:Adenosine Triphosphatases; D024701:Myosin Type V; D018995:Myosin Heavy Chains; D009218:Myosins