Magnesium, ADP, and actin binding linkage of myosin V: evidence for multiple myosin V-ADP and actomyosin V-ADP states.
The [Mg(2+)] dependence of ADP binding to myosin V and actomyosin V was measured from the fluorescence of mantADP. Time courses of MgmantADP dissociation from myosin V and actomyosin V are biphasic with fast observed rate constants that depend on the [Mg(2+)] and slow observed rate constants that are [Mg(2+)]-independent. Two myosin V-MgADP states that are in reversible equilibrium, one that exchanges nucleotide and cation slowly (strong binding) and one that exchanges nucleotide and cation rapidly (weak binding), account for the data. The two myosin V-MgADP states are of comparable energies, as indicated by the relatively equimolar partitioning at saturating magnesium. Actin binding lowers the affinity for bound Mg(2+) 2-fold but shifts the isomerization equilibrium approximately 6-fold to the weak ADP binding state, lowering the affinity and accelerating the overall rate of MgADP release. Actin does not weaken the affinity or accelerate the release of cation-free ADP, indicating that actin and ADP binding linkage is magnesium-dependent. Myosin V and myosin V-ADP binding to actin was assayed from the quenching of pyrene actin fluorescence. Time courses of myosin V-ADP binding and release are biphasic, consistent with the existence of two (weak and strong) quenched pyrene actomyosin V-ADP conformations. We favor a sequential mechanism for actomyosin V dissociation with a transition from strong to weak actin-binding conformations preceding dissociation. The data provide evidence for multiple myosin-ADP and actomyosin-ADP states and establish a kinetic and thermodynamic framework for defining the magnesium-dependent coupling between the actin and nucleotide binding sites of myosin.
Hannemann
Diane E
DE
Department of Molecular Biophysics and Biochemistry, Yale University, 260 Whitney Avenue, New Haven, Connecticut 06520, USA.
Cao
Wenxiang
W
Olivares
Adrian O
AO
Robblee
James P
JP
De La Cruz
Enrique M
EM
D000199:Actins / Q000378:metabolism*; D000205:Actomyosin / Q000737:chemistry / Q000378:metabolism*; D000244:Adenosine Diphosphate / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002645:Chickens; D007700:Kinetics; D019344:Lactic Acid; D008274:Magnesium / Q000378:metabolism*; D024701:Myosin Type V / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding
D000199:Actins; C006756:biolactin; D019344:Lactic Acid; D000244:Adenosine Diphosphate; D000205:Actomyosin; D024701:Myosin Type V; D008274:Magnesium