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• W2155391189 abstract "Myosin VI is the only pointed end-directed myosin identified and is likely regulated by heavy chain phosphorylation (HCP) at the actin-binding site in vivo. We undertook a detailed kinetic analysis of the actomyosin VI ATPase cycle to determine whether there are unique adaptations to support reverse directionality and to determine the molecular basis of regulation by HCP. ADP release is the rate-limiting step in the cycle. ATP binds slowly and with low affinity. At physiological nucleotide concentrations, myosin VI is strongly bound to actin and populates the nucleotide-free (rigor) and ADP-bound states. Therefore, myosin VI is a high duty ratio motor adapted for maintaining tension and has potential to be processive. A mutant mimicking HCP increases the rate of Pi release, which lowers theK ATPase but does not affect ADP release. These measurements are the first to directly measure the steps regulated by HCP for any myosin. Measurements with double-headed myosin VI demonstrate that the heads are not independent, and the native dimer hydrolyzes multiple ATPs per diffusional encounter with an actin filament. We propose an alternating site model for the stepping and processivity of two-headed high duty ratio myosins. Myosin VI is the only pointed end-directed myosin identified and is likely regulated by heavy chain phosphorylation (HCP) at the actin-binding site in vivo. We undertook a detailed kinetic analysis of the actomyosin VI ATPase cycle to determine whether there are unique adaptations to support reverse directionality and to determine the molecular basis of regulation by HCP. ADP release is the rate-limiting step in the cycle. ATP binds slowly and with low affinity. At physiological nucleotide concentrations, myosin VI is strongly bound to actin and populates the nucleotide-free (rigor) and ADP-bound states. Therefore, myosin VI is a high duty ratio motor adapted for maintaining tension and has potential to be processive. A mutant mimicking HCP increases the rate of Pi release, which lowers theK ATPase but does not affect ADP release. These measurements are the first to directly measure the steps regulated by HCP for any myosin. Measurements with double-headed myosin VI demonstrate that the heads are not independent, and the native dimer hydrolyzes multiple ATPs per diffusional encounter with an actin filament. We propose an alternating site model for the stepping and processivity of two-headed high duty ratio myosins. high pressure liquid chromatography N-methylanthraniloyl,2′-deoxyadenosine 5′-diphosphate N-methylanthraniloyl,2′-deoxyadenosine 5′-triphosphate myosin VI possessing two catalytic subunits, heavy meromyosin-like calmodulin phosphate-binding protein porcine myosin VI with Thr406 substituted to Ala porcine myosin VI with Thr406 substituted to Glu Myosin VI is unique among members of the myosin superfamily of molecular motors in that it moves toward the pointed ends of actin filaments as opposed to the barbed ends (1Wells A.L. Lin A.W. Chen L.Q. Safer D. Cain S.M. Hasson T. Carragher B.O. Milligan R.A. Sweeney H.L. Nature. 1999; 40: 505-508Crossref Scopus (548) Google Scholar). Although the cellular roles of myosin VI are not defined, it has been implicated in membrane trafficking and organelle transport (2Mermall V. McNally J.G. Miller K.G. Nature. 1994; 369: 560-562Crossref PubMed Scopus (111) Google Scholar, 3Buss F. Kendrick-Jones J. Lionne C. Knight A.E. Cote G.P. Luzio P.J. J. Cell Biol. 1998; 143: 1535-1545Crossref PubMed Scopus (167) Google Scholar, 4Kelleher J.F. Mandell M.A. Moulder G. Hill K.L. L'Hernault S.W. Barstead R. Titus M.A. Curr. Biol. 2000; 10: 1489-1496Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar) as well as maintaining the structural integrity of inner ear hair cells (5Avraham K.B. Hasson T. Steel K.P. Kingsley D.M. Russell L.B. Mooseker M.S. Copeland N.G. Jenkins N.A. Nat. Genet. 1995; 11: 369-375Crossref PubMed Scopus (416) Google Scholar).Native myosin VI has two “heads,” or catalytic domains, that are thought to be regulated by p21-activated kinase phosphorylationin vivo (3Buss F. Kendrick-Jones J. Lionne C. Knight A.E. Cote G.P. Luzio P.J. J. Cell Biol. 1998; 143: 1535-1545Crossref PubMed Scopus (167) Google Scholar). Although the phosphorylation site was not identified directly, it was mapped between amino acids 308 and 631 and believed to be Thr406 of the actin-binding interface, because flanking sequences have p21-activated kinase recognition sites, and phosphorylation of Thr406 would be consistent with the TEDS rule (6Bement W.M. Mooseker M.S. Cell Motil. Cytoskeleton. 1995; 31: 87-92Crossref PubMed Scopus (147) Google Scholar). All myosins have an acidic residue (Asp or Glu) at this position and are constitutively active or, in the case of Acanthamoeba myosin I, have a serine or threonine that when phosphorylated increases the ATPase rate more than 20-fold (7Albanesi J.P. Hammer J.A. Korn E.D. J. Biol. Chem. 1983; 258: 10176-10181Abstract Full Text PDF PubMed Google Scholar). Myosin I heavy chain kinases are p21-activated kinase homologues (8Brzeska H. Szczepanowska J. Hoey J. Korn E.D. J. Biol. Chem. 1996; 271: 27056-27062Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 9Wu C. Lee S.F. Furmaniak-Kazmierczak E. Cote G.P. Thomas D.Y. Leberer E. J. Biol. Chem. 1996; 271: 31787-31790Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). Mutagenesis of Acanthamoeba myosin I demonstrates that the phosphorylated and unphosphorylated states are mimicked by replacement of the phosphorylatable threonine with a glutamate or alanine, respectively (10Wang Z.Y. Wang F. Sellers J.R. Korn E.D. Hammer J.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15200-15205Crossref PubMed Scopus (36) Google Scholar).The molecular mechanism by which myosin VI achieves its reverse directionality is not known, but it might be linked to unique aspects of the converter domain, the structural element between the motor and light chain binding domains (1Wells A.L. Lin A.W. Chen L.Q. Safer D. Cain S.M. Hasson T. Carragher B.O. Milligan R.A. Sweeney H.L. Nature. 1999; 40: 505-508Crossref Scopus (548) Google Scholar). In this study, we define the kinetic mechanism of the actomyosin VI ATPase cycle to ascertain if pointed end-directed motility requires unique biochemical adaptations. In addition, to determine whether myosin VI is regulated by heavy chain phosphorylation and to define the molecular basis of the regulation, we characterized the kinetics of myosin VI with a glutamate or alanine substitution at Thr406. As demonstrated for other unconventional myosins (11Ostap E.M. Pollard T.D. J. Cell Biol. 1996; 32: 1053-1060Crossref Scopus (82) Google Scholar, 12De La Cruz E.M. Wells A.L. Rosenfeld S.S. Ostap E.M. Sweeney H.L. Proc. Natl. Acad. Sci. 1999; 96: 13726-13731Crossref PubMed Scopus (352) Google Scholar, 13De La Cruz E.M. Sweeney H.L. Ostap E.M. Biophys. J. 2000; 79: 1524-1529Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 14De La Cruz E.M. Wells A.L. Sweeney H.L. Ostap E.M. Biochemistry. 2000; 39: 14196-14202Crossref PubMed Scopus (74) Google Scholar), kinetic characterization helps define the biological functions and degree of processivity of myosin VI.RESULTSUnless specified, all experimental measurements were made with subfragment 1-like myosin VI consisting of the motor domain and associated light chain. “Myosin VI-HMM” is used when referring to the two-headed construct.Steady-state ATPase of Myosin VIThe steady-state MgATPase activities of myosin VI-T406E and T406A are activated from <0.1 s−1 (v o) to ∼8–9 s−1 (V max) by actin filaments (Fig.2, Table I). TheK ATPase of myosin VI-T406E is ∼3 µm. Myosin VI-T406A has a much higherK ATPase of ∼18 µm. Therefore, actin filaments activate the ATPase of myosin VI-T406E and T406A ≥ 100-fold but with dramatically different K ATPasevalues (Fig. 2, Table I).Table ISteady-state ATPase parameters for myosin VIT406ET406AT406E (HMM)V max (sec−1head−1)1-aMaximum steady-state ATPase rate in the presence of saturating actin filaments.8.3 (±0.2)1-bUncertainties represent standard errors in the best fits of the data.9.1 (±0.8)3.3 (±0.1)K ATPase(µm)1-cActin filament concentration at half-maximum activation of steady-state ATPase.2.8 (±0.3)17.6 (±2.0)0.6 (±0.1)v o (sec−1head−1)1-dSteady-state ATP turnover rate in the absence of actin filaments.1-eDetermined using the NADH-coupled assay.<0.1<0.1∼0.1v o (sec−1head−1)1-dSteady-state ATP turnover rate in the absence of actin filaments.1-fMeasured using PiBiP at 100 µm MgATP.0.04 (±0.01)0.04 (±0.01)N.D.1-gNot determined.Assay conditions were: 50 mm KCl, 1 mmMgCl2, 1 mm EGTA, 1 mm dithiothreitol, 2 mm MgATP, 10 mm imidazole (pH 7.0) at 25 °C.1-a Maximum steady-state ATPase rate in the presence of saturating actin filaments.1-b Uncertainties represent standard errors in the best fits of the data.1-c Actin filament concentration at half-maximum activation of steady-state ATPase.1-d Steady-state ATP turnover rate in the absence of actin filaments.1-e Determined using the NADH-coupled assay.1-f Measured using PiBiP at 100 µm MgATP.1-g Not determined. Open table in a new tab Myosin VI-T406E-HMM (two catalytic subunits or heads) has aV max of 3.3 s−1 head−1and K ATPase of ∼0.6 µm (Fig. 2, Table I). A reduction in V max andK ATPase suggests the heads of the myosin VI dimer do not act independently (see “Discussion”).The actin concentration dependence of wild-type (Thr406) myosin VI ATPase activity does not fit a hyperbola well (data not shown) but seems to be the sum of two hyperbolas withK ATPase values of ∼3 and ∼18 µm. This suggests a mixture of phosphorylated and dephosphorylated myosin VI is purified from baculovirus as demonstrated for myosin I (10Wang Z.Y. Wang F. Sellers J.R. Korn E.D. Hammer J.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15200-15205Crossref PubMed Scopus (36) Google Scholar). The V max of wild-type myosin VI was ∼6 s−1 (data not shown). TheV max of wild-type myosin VI-HMM is ∼2.5 s−1 head−1.In the absence of an ATP-regenerating system, the maximum steady-state ATPase rates of wild-type and mutant myosin VI are slow (V max ≤ 1 s−1, data not shown, see also Ref. 1Wells A.L. Lin A.W. Chen L.Q. Safer D. Cain S.M. Hasson T. Carragher B.O. Milligan R.A. Sweeney H.L. Nature. 1999; 40: 505-508Crossref Scopus (548) Google Scholar) because of product inhibition by ADP (13De La Cruz E.M. Sweeney H.L. Ostap E.M. Biophys. J. 2000; 79: 1524-1529Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar).Myosin VI Binding to Actin Filaments by Pyrene FluorescenceAs demonstrated for other myosins, strongly bound myosin VI (AM and AM.ADP in SchemeFS1) quenches ∼75% of the pyrene-actin fluorescence. Time courses of the reduction in fluorescence intensity after mixing myosin VI-T406E (± ADP) or myosin VI-T406A (± ADP) with pyrene-actin filaments all follow single exponentials (Fig.3A) with no distinguishable lag phase (12De La Cruz E.M. Wells A.L. Rosenfeld S.S. Ostap E.M. Sweeney H.L. Proc. Natl. Acad. Sci. 1999; 96: 13726-13731Crossref PubMed Scopus (352) Google Scholar, 21Taylor E.W. J. Biol. Chem. 1991; 266: 294-302Abstract Full Text PDF PubMed Google Scholar). The myosin and actin concentrations were adjusted such that actin was ∼10 times the myosin concentration and pseudo first-order requirements were fulfilled. The observed rates (k obs) depend linearly on the actin filament concentration over the range examined (Fig. 3 B). The data were modeled as simple bimolecular reactions according to Schemes FS2andFS3,Figure FS1View Large Image Figure ViewerDownload (PPT)Figure 3Kinetics of myosin VI binding to actin filaments. A, time course of fluorescence quenching after mixing 1.4 µm pyrene actin with 0.15 µm myosin VI-T406E (curve a), 0.15 µm myosin VI-T406E (curve b) in the presence of 500 µm MgADP or buffer alone (curve c). Thesmooth lines through the data (curves a andb) are the best fits to single exponential functions (y = A e−kt +c, where A is the amplitude, k is the observed rate, t is time, and the start is A +c) with rates of 8.9 s−1 (curve a) or 3.0 s−1 (curve b). The data represented are raw, unaveraged, and unfiltered. Typically 4–7 transients were averaged before fitting. Only the first second of the acquired time course is shown. B, concentration dependence of the observed rate (k obs) of actin filament binding to myosin VI-T406E (●), myosin VI-T406E with bound ADP (▴), myosin VI-T406A (○), and myosin VI-T406A with bound ADP (▵). C, time course of fluorescence increase after mixing 0.5 µmactomyosinVI-T406A in the presence (curve a) or absence (curve b) of 500 µm MgADP with 40 µm unlabeled actin filaments. Only the first 400 s of the time courses are shown for clarity. The smooth linesare the best fits of the data to single exponentials (y= A (1 − e−kt) + start, whereA is the amplitude, k is the observed rate, andt is time). The rates were 0.04 s−1(curve a) or 0.002 s−1 (curve b) for this preparation.View Large Image Figure ViewerDownload (PPT)Figure FS2View Large Image Figure ViewerDownload (PPT)Figure FS3View Large Image Figure ViewerDownload (PPT)where the * indicates high fluorescence (i.e.unquenched) of pyrene actin. The apparent second-order rate constants (k −6 and k −10) for myosin VI binding to actin filaments were obtained from the slopes of the lines and are summarized in TableII.Table IIThe rate and equilibrium constants of the actomyosin VI-11Q ATPase cycleT406ET406AActin binding2-aPyrene actin.k −6 (µm−1s−1)5.4 (±0.2)6.8 (±0.2)k +6(s−1)0.005 (±0.004)2-b, uncertainties represent one S.D. with n = 3 from two different preparations (b), n = 4 from two preps (c), orn = 8 from two preps (d),0.004 (±0.002)2-b, uncertainties represent one S.D. with n = 3 from two different preparations (b), n = 4 from two preps (c), orn = 8 from two preps (d),K 6(nm)0.9 (±0.7)0.6 (±0.3)k −10 (µm−1s−1)0.9 (±0.1)1.5 (±0.1)k −10 (µm−1s−1), HMM2.0 (±0.2)k +10(s−1)0.06 (±0.03)c0.07 (±0.03)dK 10(nm)67 (±34)47 (±21)K 9(µm)2-e, PiBiP;32 (±6)39 (±27)ATP binding and hydrolysisK 1′k +2′ (µm−1s−1)2-aPyrene actin.0.018 (±0.001)0.015 (±0.001)k +2′ (s−1)2-aPyrene actin.>250>250K 1(k +2 +k −2) (µm−1s−1) 2-f, mantATP;0.27 (±0.04)0.14 (±0.04)k −2(s−1) 2-f, mantATP;3.9 (±0.3)4.0 (±0.3)K 32-g, [γ-32P]-ATP.K 3 = (k +3/k −3);≥1.4 (±0.1)2-h, uncertainties inK 3 are S.D. with n = 3;≥1.5 (±0.5)2-h, uncertainties inK 3 are S.D. with n = 3;Pi release 2-e, PiBiP;k +4′ (s−1)89 (±10)34 (±15)k +4(s−1)0.04 (±0.01)0.04 (±0.01)ADP binding 2-i, mantADP;k −5 (µm−1s−1)1.06 (±0.10)0.26 (±0.04)k +5(s−1)6.4 (±0.1)5.6 (±0.2)K 5(µm)6.0 (±0.6)21.5 (±3.3)k −5′ (µm−1s−1)0.60 (±0.10)0.18 (±0.03)k +5′ (s−1)5.6 (±0.1)5.4 (±0.2)k +5′ (s−1), HMM 2-j, mantADP was competed with excess unlabelled ATP.7.0 (±0.4)K 5′ (µm)8.8 (±1.4)30 (±5)Assay conditions were: 50 mm KCl, 1 mmMgCl2, 1 mm EGTA, 1 mm DTT, 10 mm imidazole, pH 7.0, 25 °C.2-a Pyrene actin.2-b–d , uncertainties represent one S.D. with n = 3 from two different preparations (b), n = 4 from two preps (c), orn = 8 from two preps (d),2-e , PiBiP;2-f , mantATP;2-g , [γ-32P]-ATP.K 3 = (k +3/k −3);2-h , uncertainties inK 3 are S.D. with n = 3;2-i , mantADP;2-j , mantADP was competed with excess unlabelled ATP. Open table in a new tab Myosin VI dissociation from pyrene actin was measured by competition after mixing an equilibrated mixture of pyrene-actomyosin VI (± ADP) with an 80-fold excess of unlabeled actin filaments (Fig.3 C). The rate-limiting step, and therefore the step measured, is dissociation of myosin VI bound to pyrene actin. In the presence and absence of ADP, time courses of fluorescence enhancements follow single exponentials and yield the apparent dissociation rates (k +6 and k +10) for actomyosin VI dissociation (Table II). The apparent actomyosin VI affinities (K 6 and K 10) were determined from the ratio of the rate constants (Table II).Phosphorylation at Thr406 has minimal effects (<2-fold and within the range of uncertainty) on the rates and affinities of actin-filament binding in the strongly bound (AM and AM.ADP) states (Table II).ADP weakens the affinity of myosin VI for actin filaments ∼80-fold independent of phosphorylation at Thr406; the rate of binding to actin is reduced ∼4–6-fold, and the rate of dissociation from actin is ∼10–20 times more rapid (Table II).ATP-induced Population of the Weakly Bound StatesThe fluorescence of pyrene actin was used to monitor formation of the weak binding states of myosin VI after mixing with ATP (Fig.4). Concentrations lower than theK ATPase (Table I) were used to ensure ATP-induced dissociation of myosin VI from actin as confirmed by light scattering (data not shown). The addition of MgATP to 0.5 µm strongly bound (i.e. quenched) pyrene-actomyosin VI increases the fluorescence to that of pyrene actin alone. The time courses follow single exponentials (Fig. 4,A and B) with rates that depend linearly on the MgATP concentration (Fig. 4 C). As for all other characterized myosins, the mechanism of ATP-induced fluorescence enhancement was modeled as a two-step binding reaction.Figure 4MgATP induced population of weakly bound actomyosin VI states. A, time course of fluorescence increase after mixing 500 (curve a), 300 (curve b), 100 (curve c), or 0 µm (curve d) MgATP with 0.5 µm pyrene-actomyosin VI-T406E. Thesmooth lines are the best fits to single exponentials with the following rates (k obs): curve a, 6.3 s−1; curve b, 3.9 s−1;curve c, 1.7 s−1. B, time course of fluorescence change after mixing 12 mm MgATP with 0.2 µm myosin VI-T406A bound to pyrene actin at a 1:1 molar ratio. The smooth line is the best fit to a single exponential with a rate (k obs) of 161 s−1. C, ATP concentration dependence of thek obs for myosin VI-T406E (●) or myosin VI-T406A (○). The apparent second-order association rate constants for ATP binding to actomyosin VI (K 1′k +2′) determined from the slopes of the lines are presented in Table II.View Large Image Figure ViewerDownload (PPT)where AM(ATP) is the quenched collision complex in rapid equilibrium (K 1′) with free nucleotide that isomerizes (k +2′) to the high fluorescence A*M.ATP. At saturating ATP (>5 mm), 100% of the fluorescence is recovered, demonstrating thatk +2′ ≫ k −2′ and ATP binding can be considered to be essentially irreversible.The association rate constants for MgATP binding to actomyosin VI (K 1′k +2′) obtained from the initial slopes of the lines are 18.2 (± 0.2) mm−1 s−1 for T406E and 15.2 (± 0.4) mm−1 s−1 for T406A (TableII). The maximum rate of ATP-induced dissociation (k +2′ + k −2′) for both myosin VI mutants is >250 s−1. The equilibrium constant for rapid ATP binding is very weak (1/K 1′ ≫ 14 mm) for both myosin VI mutants, suggesting that either ATP dissociates rapidly from the collision complex (rapidk −1′) or the nucleotide binding site is not readily accessible (slow k +1′, see “Discussion”). From the amplitudes of the transients we estimate apparent K m values of ∼300 µm for both myosin VI mutants.Although we did not correct for the changes in the ionic strength of the buffer resulting from the addition of mm MgATP, inclusion of an additional 50 mm KCl (corresponding to ∼12 mm MgATP assuming a net charge of −2 at pH 7.0) slowed the rate of T406A dissociation at 8 mm MgATP from 113 s−1 to 91 s−1 (data not shown). Therefore, we are confident that the dependence of the observed rate on [ATP] is linear over the range examined and that changes in ionic strength resulting from the addition of MgATP are not preventing the detection of a biphasic dependence (i.e. saturation) of the observed rates.At rates >50 s−1, a slow component with a rate of 7–9 s−1 could be resolved in the transients (data not shown). The amplitude (2–5%) of this phase was variable and could be accounted for by the fraction of ADP in the solution and the experimentally determined rates of ATP binding, ADP binding (presented below), and ADP release (presented below), suggesting that the slow component results from residual ADP in the ATP (22Geeves M.A. Perreault-Micale C. Coluccio L.M. J. Biol. Chem. 2000; 275: 21624-21630Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar).MgATP Binding to Myosin VI in the Absence of Actin by mantATP FluorescenceThe fluorescent nucleotide 2′-deoxy-mantATP (λex = 365 nm) was used to monitor MgATP binding to myosin VI in the absence of actin (Fig.5). Time courses of fluorescence enhancement after mixing mantATP with myosin VI do not follow single exponentials but can be fitted to double exponential functions (Fig.5 A). The rate of the fast phase depends on the mantATP concentration for both mutants (Fig. 5 B). The second-order association rate constants for ATP binding obtained from the slopes of the lines are 0.27 µm−1 s−1 for T406E and 0.14 µm−1 s−1 for T406A. The intercepts give dissociation rate constants of ∼4 s−1 for both mutants (Table II).Figure 5Kinetics of mantATP binding to myosin VI in the absence of actin. A, time course of fluorescence increase after mixing 2.1 µm 2′-deoxy-mantATP with 0.3 µm myosin VI-T406E in the absence of actin. The transient is the average of three individual traces. The smooth lineis the best fit of the data to a double exponential function with rates of 4.6 and 0.2 s−1. B, dependence of the fast phase k obs on [mantATP]. ●, myosin VI-T406E; ○, myosin VI-T406A. The rate constants for mantATP binding are presented in Table II.View Large Image Figure ViewerDownload (PPT)We could not accurately resolve the slow phase because of interference from photobleaching over long time scales but the rates were ∼0.2–0.4 s−1 and, given the uncertainties in the values, difficult to conclude if they depend on the nucleotide concentration. The source of the slow component is unknown, but it may represent a fraction of myosin that binds ATP (but not ADP, see below and Fig. 6A) slowly or an isomerization of myosin and/or nucleotide after binding. We did not investigate this component because it is not likely to be a relevant pathway in the actin-activated ATPase cycle; the rate is much slower than the steady-state turnover rate (Fig. 2 B).Figure 6Kinetics of mantADP binding to myosin VI and actomyosin VI. A, time course of fluorescence increase after mixing 6.1 µm 2′-deoxy-mantADP with 0.4 µm myosin VI-T406A in the absence of actin. The transient is of raw, unaveraged, and unfiltered data. The smooth linethrough the data is the best fit to a single exponential with a rate of 7.2 s−1. B, dependence ofk obs on [mantADP]. ▴, myosin VI-T406E; ●, actomyosin VI-T406E; ▵, myosin VI-T406A; ○, actomyosin VI-T406A. The rate and equilibrium constants for mantADP binding are presented in Table II. C, time course of mantADP dissociation from actomyosin VI-T406E. An equilibrated mixture of 2′-deoxy-mantADP (40 µm) and actomyosin VI (1 µm) was mixed with 2 mm unlabeled ADP. The smooth line through the data is the best fit to an exponential with a rate of 5.6 s−1.View Large Image Figure ViewerDownload (PPT)In the presence of actin the rates of mantATP binding were too slow (see Fig. 4 C, MgATP binding to pyrene actomyosin) to measure accurately or to distinguish the fast and slow phases of the reaction.There are no detectable changes in tryptophan fluorescence of either myosin VI mutant with ATP (or ADP) binding or ATP hydrolysis (data not shown). Similarly, there are no detectable changes in mantATP (or mantADP) fluorescence (λex = 295 nm) resulting from energy transfer via tryptophans to the bound mant nucleotides (data not shown). Tryptophans corresponding to chicken skeletal muscle myosin Trp510 and Trp595are absent from myosin VI (Val504 and Phe594, respectively), suggesting that either or both of these residues contribute to the fluorescence changes observed with other myosins. The Trp510 equivalent (Trp512) monitors the structural change preceding ATP hydrolysis in smooth muscle myosin II (23Yengo C.M. Chrin L.R. Rovner A.S. Berger C.L. J. Biol. Chem. 2000; 18: 25481-25487Abstract Full Text Full Text PDF Scopus (54) Google Scholar).ADP-Pi Burst and the Equilibrium Constant for ATP Hydrolysis (K3)We measured the equilibrium constant for ATP hydrolysis (K 3 =k +3/k −3) from the amplitude of the ADP-Pi burst (B = [ADP-Pi]/[myosin]). In the absence of actin, both myosin VI mutants display a burst amplitude of ∼0.6 ADP-Pi/myosin (T406E = 0.58 and T406A = 0.60) after mixing with 200 µm ATP. Using the relationB = K 3/(1 +K 3), we calculate a K 3 of 1.4 (± 0.1) for myosin VI-T406E and 1.5 (± 0.2) for myosin VI-T406A (Table II). It is important to clarify that this represents a lower limit for K 3, because ATP binding appears to be reversible (Fig. 5 B, see “Discussion”), and the measured burst amplitude may be low as a result of rapid ATP dissociation. We could not get a reliable signal at higher [ATP].The slow rate of ATP binding to actomyosin VI (K 1′k +2′, Fig. 4, TableII) precludes measurement of the ADP-Pi burst in the presence of actin.ADP Binding and Dissociation by mantADP FluorescenceThe fluorescence enhancement of 2′-deoxy-mantADP upon binding to myosin was used to measure the affinity and rates of ADP binding to myosin VI (Fig. 6, Table II). Time courses of fluorescence enhancements after mixing mantADP with myosin VI and actomyosin VI all follow single exponentials (Fig. 6 A) with rates that depend linearly on the concentration of nucleotide (Fig.6 B). Therefore, mantADP binding was modeled as a simple bimolecular reaction according to SchemesFS5andFS6,Figure FS5View Large Image Figure ViewerDownload (PPT)Figure FS6View Large Image Figure ViewerDownload (PPT)where the * indicates high mantADP fluorescence, yielding the apparent second-order rate constants for mantADP binding (k −5 and k −5′) from the slopes of the lines (Table II). The intercepts deviate from the origin and reflect the dissociation rates (k +5′ and k +5) as confirmed by direct measurement (Fig. 6 C).The rate of mantADP release from actomyosin VI-T406E and -T406A is ∼6 s−1 (Fig. 6 C, Tables I and 2), which approximates the steady-state ATPase rate of both mutants, suggesting that ADP release is rate-limiting in the presence of actin. Myosin VI-T406E-HMM dissociates mantADP at approximately the same rate (7.0 ± 0.4 s−1, Table II). For other characterized myosins (e.g. see Ref. 12De La Cruz E.M. Wells A.L. Rosenfeld S.S. Ostap E.M. Sweeney H.L. Proc. Natl. Acad. Sci. 1999; 96: 13726-13731Crossref PubMed Scopus (352) Google Scholar), mantADP release from actomyosin is 1.5–2-fold slower than unlabeled ADP.Actin binding has minimal (<2-fold reduction) effects on the rates and affinities of mantADP binding (Table II). Dissociation (Fig.6 C) occurs at ∼6 s−1 from both myosin-VI mutants in the presence (k +5′) and absence (k +5) of actin. The association rate constants of mantADP are reduced only slightly (<2-fold) by binding to actin.Myosin VI-T406E binds mantADP with a 3–4-fold higher affinity (K 5 and K 5′) than does T406A because of a more rapid association rate constant both in the presence (k −5) and absence (k −5′) of actin filaments (Table II). Therefore, actomyosin VI binds MgADP more tightly than MgATP (K m values for ATP ∼ 300 µm): ∼30–40 times more tightly for T406E and 10–15 times more tightly for T406A.Pi ReleaseTime courses of Pi release after mixing myosin VI-ADP-Pi with actin filaments show a rapid exponential burst phase followed by a slow linear phase for both myosin VI mutants (Fig. 7, Aand B). The burst corresponds to the first turnover of Pi release after actin binding, and the slow linear phase reflects steady-state ATPase activity.Figure 7Rate of Pi release from actomyosin VI. A, time course of transient Pirelease from myosin VI-T406E-ADP-Pi after mixing with 20 (curve a), 9 (curve b), or 0 µm(curve c) actin filaments. B, time course of transient Pi release from myosin VI-T406A-ADP-Pi after mixing with 20 (curve a), 9 (curve b), or 0 µm (curve c) actin filaments. Note that the time scales between A andB are different. C, actin filament concentration dependence of the Pi release burst rate. Final concentrations at t = 0 were 1.5 µmmyosin VI, 4.5 µm PiBiP, 100 µmMgATP, and the indicated actin filament concentrations. TheV max and K ATPase values obtained from the linear phase of these curves are lower than those obtained with the NADH assays (Fig. 2) because of the low ATP concentration (100 µm versus 2 mm) and ADP inhibition without an ATP regenerating system.View Large Image Figure ViewerDownload (PPT)The burst was measured without interference from the linear phase by including ADP (1 mm) with actin (data not shown). ADP competes with ATP and limits myosin to a single ATP turnover. There is no burst phase for either mutant in the absence of actin (curve c in Fig. 7, A and B), thus Pirelease is rate-limiting (k +4 ∼ 0.04 s−1).The Pi release burst rate depends hyperbolically on the actin filament concentration for both myosin VI mutants (Fig.7 C) and was therefore modeled as a two-step binding r" @default.
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• W2155391189 title "Kinetic Mechanism and Regulation of Myosin VI" @default.
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