Kinetic analyses of a truncated mammalian myosin I suggest a novel isomerization event preceding nucleotide binding

J Biol Chem. 2000 Jul 14;275(28):21624-30. doi: 10.1074/jbc.M000342200.

Abstract

MI(1IQ) is a complex of calmodulin and an epitope-tagged 85-kDa fragment representing the amino-terminal catalytic motor domain and the first of 6 calmodulin-binding IQ domains of the mammalian myosin I gene, rat myr-1 (130-kDa myosin I or MI(130)). We have determined the transient kinetic parameters that dictate the ATP hydrolysis cycle of mammalian myosin I by examining the properties of MI(1IQ). Transient kinetics reveal that the affinity of MI(1IQ) for actin is 12 nm. The ATP-induced dissociation of actin-MI(1IQ) is biphasic. The fast phase is dependent upon [ATP], whereas the slow phase is not; both phases show a Ca(2+) sensitivity. The fast phase is eliminated by the addition of ADP, 10 micrometer being required for half-saturation of the effect in the presence of Ca(2+) and 3 micrometer ADP in the absence of Ca(2+). The slow phase shares the same rate constant as ADP release (8 and 3 s(-)(1) in the presence and absence of Ca(2+), respectively), but cannot be eliminated by decreasing [ADP]. We interpret these results to suggest that actin-myosin I exists in two forms in equilibrium, one of which is unable to bind nucleotide. These results also indicate that the absence of the COOH-terminal 5 calmodulin binding domains of myr-1 do not influence the kinetic properties of MI(130) and that the Ca(2+) sensitivity of the kinetics are in all likelihood due to Ca(2+) binding to the first IQ domain.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Actins / metabolism
  • Adenosine Diphosphate / metabolism
  • Adenosine Triphosphate / metabolism
  • Animals
  • Binding Sites
  • Calcium / metabolism
  • Calmodulin / metabolism
  • Isomerism
  • Kinetics
  • Liver / metabolism
  • Models, Molecular
  • Muscle, Skeletal / metabolism
  • Myosin Type I*
  • Myosins / chemistry*
  • Myosins / metabolism*
  • Protein Conformation
  • Rats
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / metabolism
  • Sequence Deletion

Substances

  • Actins
  • Calmodulin
  • Myo1b protein, rat
  • Recombinant Proteins
  • Adenosine Diphosphate
  • Adenosine Triphosphate
  • Myosin Type I
  • Myosins
  • Calcium