Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.6.4.1 (myosin ATPase)
1,140 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

G-actin has been nitrated with tetranitromethane in conditions that lead to the modification of one tyrosine residue. The reactive residue was found by earlier workers to be Tyr-69. The nitrated actin is conformationally similar to native G-actin, as judged by sedimentation velocity and circular dichroism analysis. A small proportion only is in the form of covalently linked dimers and trimers. The nitrated G-actin will polymerise to form filaments, indistinguishable in the electron microscope from those of native F-actin, but the polymerisation process is slower. Reduction of the nitrophenol group to the corresponding aminophenol leaves the properties of the protein in respect of polymerisation unchanged. When a dansyl group is introduced at the same point, however, the ability of the actin to polymerise is lost. The nitrated actin and its reduced counterpart will also bind heavy meromyosin, and the characteristic arrowhead formation of the bound molecules along the filaments can be seen in the electron microscope. Neither of the modified F-actins, however, significantly activates or inhibits the myosin ATPase activity. The fluorescence of nitrated actin is strongly quenched through the presence of the nitrophenol chromophore. In soluble complexes with heavy meromyosin the fluorescence is indistinguishable from the sum of the separate contributions of the two protein components. There is thus no measurable excitation transfer between any tryptophan residues on the myosin heads, such as that inferred to be present in the ATPase site, and the nitrotyrosine in position 69 of the actin sequence. Implications of this observation are considered in relation to the different interaction sites in myosin and in actin. The activation of heavy meromyosin ATPase by copolymers containing actin and nitroactin in different proportions has been measured, and is not proportional to the fraction of native actin. The results are consistent with the view that the function of actomyosin depends on the interaction of the myosin heads with more than one actin subunit.
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PMID:Effects of specific chemical modification of actin. 12 59

Myosin was purified from the flight muscles of a flying (pigeon) and a nonflying (fowl) bird. Ki (ADP) of myosin ATPase of pigeon is higher, but the Km (ATP) is lower than that of fowl. The specific activity (mumole of Pi liberated/min/mg protein) is higher for the fowl. A0.5 (CaCl2) of myosin of both pigeon and fowl is similar. However, the two proteins differ in their interactions with ADP, ATP and p-chloromercuribenzoate. The two proteins have the same tyrosine, tryptophan and sulfhydryl contents. The electrophoretic patterns of the two myosins on SDS-polyacrylamide gels are different. These studies show significant molecular differences in the myosin derived from the flight muscles of a flying (pigeon) and a nonflying (fowl) bird.
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PMID:Comparative studies on myosin ATPase of a flying and nonflying bird. 15 58

The reactive thiol Cys-697 (SH2) in myosin ATPase was labeled with a fluorescent analog of maleimide, 2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid (MIANS) (Hiratsuka, T. (1992) J. Biol. Chem. 267, 14941-14948). Although the tryptophan fluorescence of myosin subfragment-1 (S-1) was slightly affected by incorporation of the MIANS fluorophore, the tryptophan fluorescence of the resultant S-1 derivative (MIANS-S-1) was enhanced by ATP in a manner similar to that of unlabeled S-1. The quenching of tryptophan fluorescence of MIANS-S-1 was shown to result from a transfer of the excitation energy from tryptophanyl residue(s) to the MIANS fluorophore attached to SH2, which absorbed and fluoresced maximally at 325 and 418 nm, respectively. The energy transfer measurements were performed in the presence of acrylamide and compared to those performed in the absence of the quencher. The energy transfer efficiencies were found to be unaltered by acrylamide, indicating that the observed fluorescence energy transfer is originated exclusively from the tryptophanyl residue(s) that are not affected by acrylamide, i.e. the ATP-sensitive tryptophanyl residue(s) of S-1 (Torgerson, P. M. (1984) Biochemistry 23, 3002-3007). The distance between the tryptophanyl residue(s) and Cys-697 was calculated to be 27 A assuming a single donor-acceptor pair. Trp-510 is proposed to be one of the ATP-sensitive tryptophanyl residues.
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PMID:Spatial proximity of ATP-sensitive tryptophanyl residue(s) and Cys-697 in myosin ATPase. 138 83

Inhibition of the myosin subfragment 1 (S-1) ATPase activity by beryllium fluoride was studied directly in the presence of MgATP and following preincubation of samples with MgADP. In both cases, the rates of inhibition were very slow, with kapp = 0.5 and 58 M-1 s-1, respectively, in analogy to the rates of inhibition of myosin ATPase by vanadate [Goodno, C. C. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 2620-2624]. The very different rates of inhibition in the presence of MgATP and on preincubation with MgADP suggested that beryllium fluoride binds to the M.ADP state of myosin. The slow inhibition rates and the nonlinear dependence of the observed rates on beryllium fluoride concentration were consistent with a two-step inhibition process involving a rapid binding equilibrium to yield a collisional complex, M.ADP.BeF3-, and its slow isomerization into M++.ADP.BeF3-. A third, much slower, step was required to account for the conversion of the stable M++.ADP.BeF3- to a virtually irreversibly inhibited complex. Kinetic description of the inhibition pathway was derived from the observed rates of inhibition of myosin ATPase, information on the binding of beryllium fluoride to M.ADP, and measurements of epsilon ADP chase from M++.epsilon ADP.BeF3-. The isomerization rate and equilibrium constants were 1.4 x 10(-2) s-1 and 50, respectively, and the overall binding constant of beryllium fluoride to M.ADP was 5 x 10(5) M-1. The inhibitory complex showed a 16% enhancement to tryptophan fluorescence of S-1 and a reduced quenching of epsilon ADP by acrylamide. It is concluded that M++.ADP.BeF3- is analogous to the M++.ADP.Vi and M**.ADP.Pi states of myosin.
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PMID:Inhibition of myosin ATPase by beryllium fluoride. 153 58

The binding of Ca(2+)- and Ba(2+)-calmodulin to caldesmon and its functional consequence was investigated with three different calmodulin mutants. Two calmodulin mutants have pairs of cysteine residues substituted and oxidized to a disulphide bond in either the N- or C-terminal lobe (C41/75 and C85/112). The third mutant has phenylalanine-92 replaced by alanine (F92A). Binding measurements in the presence of Ca2+ by separation on native gels and by carbodiimide-induced cross-linking showed a lower affinity for caldesmon in all the mutants. When Ca2+ was replaced by Ba2+ the affinity of calmodulin for caldesmon was further reduced. The ability of Ca(2+)-calmodulin to release caldesmon's inhibition of the actin-tropomyosin-activated myosin ATPase was virtually abolished by mutation of phenylalanine-92 to alanine or by replacing Ba2+ for Ca2+ in native calmodulin. Both cysteine mutants retained their functional ability, but the increased concentration needed for 50% release of caldesmon inhibition reflected their decreased affinity. Ca2+ -calmodulin produced a broadening in the signals of the NMR spectrum of the 10 kDa Ca(2+)-calmodulin-binding C-terminal fragment of caldesmon arising from tryptophans -749 and -779 and caused an enhancement of maximum tryptophan fluorescence of 49% and a 16 nm blue shift of the maximum. Ca(2+)-calmodulin F92A produced a change in wavelength of 4 nm but no change in maximum, whereas Ca(2+)-calmodulin C41/75 binding produced a decrease in fluorescence with no shift of the maximum. We conclude that functional binding of Ca(2+)-calmodulin to caldesmon requires multiple interaction sites on both molecules. However, some structural modification in calmodulin does not abolish the caldesmon-related functionality. This suggests that various EF hand proteins can substitute for the calmodulin molecule.
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PMID:Multiple-sited interaction of caldesmon with Ca(2+)-calmodulin. 868 82

We have used isotope-edited nuclear magnetic resonance spectroscopy, binding studies, and ATPase activity assays to investigate the interaction with F-actin of the 10 kDa C-terminal 658C fragment of chicken gizzard caldesmon and two site-directed mutants of this fragment. Simultaneous dual-sited contacts with F-actin are observed for the segments of the 658C sequence flanking tryptophan residues 692 and 722. Competition experiments showed that both 658C contacts with actin are displaced by substoichiometric concentrations of the short inhibitory region of troponin-I indicative of different binding sites on actin for these regions of troponin-I and caldesmon. Substitution of caldesmon serine-702 by aspartic acid within the spacer region linking the two actin contacts of 658C led to weaker binding but with retention of equivalent affinity for each interaction site. Differential binding affinity of the two sites was achieved by replacement of the sequence Glu691-Trp-Leu-Thr-Lys-Thr696 by Pro-Gly-His-Tyr-Asn-Asn. Consistent with these data, the concentration of this Cg1 mutant required to achieve 50% inhibition of actin-tropomyosin-activated myosin ATPase was 4-fold greater than found for the 658C fragment. Although calmodulin binding to Cg1 was observed, calmodulin proved ineffective in relieving the inhibition induced by the binding of this mutant to actin. These results are discussed in light of the actin contacts which are involved in the inhibitory activity possessed by different regions of the C-terminus of caldesmon.
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PMID:Structure-activity studies of the regulatory interaction of the 10 kilodalton C-terminal fragment of caldesmon with actin and the effect of mutation of caldesmon residues 691-696. 948 78

Magnesium (Mg2+) is the physiological divalent cation stabilizing nucleotide or nucleotide analog in the active site of myosin subfragment 1 (S1). In the presence of fluoride, Mg2+ and MgADP form a complex that traps the active site of S1 and inhibits myosin ATPase. The ATPase inactivation rate of the magnesium trapped S1 is comparable but smaller than the other known gamma-phosphate analogs at 1.2 M-1 s-1 with 1 mM MgCl2. The observed molar ratio of Mg/S1 in this complex of 1.58 suggests that magnesium occupies the gamma-phosphate position in the ATP binding site of S1 (S1-MgADP-MgFx). The stability of S1-MgADP-MgFx at 4 degrees C was studied by EDTA chase experiments but decomposition was not observed. However, removal of excess fluoride causes full recovery of the K+-EDTA ATPase activity indicating that free fluoride is necessary for maintaining a stable trap and suggesting that the magnesium fluoride complex is bonded to the bridging oxygen of beta-phosphate more loosely than the other known phosphate analogs. The structure of S1 in S1-MgADP-MgFx was studied with near ultraviolet circular dichroism, total tryptophan fluorescence, and tryptophan residue 510 quenching measurements. These data suggest that S1-MgADP-MgFx resembles the M**.ADP.Pi steady-state intermediate of myosin ATPase. Gallium fluoride was found to compete with MgFx for the gamma-phosphate site in S1-MgADP-MgFx. The ionic radius and coordination geometry of magnesium, gallium and other known gamma-phosphate analogs were compared and identified as important in determining which myosin ATPase intermediate the analog mimics.
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PMID:Inhibition of myosin ATPase by metal fluoride complexes. 1008 41

Steady-state and time-resolved fluorescence measurements were performed on a Dictyostelium discoideum myosin II motor domain construct retaining a single tryptophan residue at position 501, located on the relay loop. Other tryptophan residues were mutated to phenylalanine. The Trp-501 residue showed a large enhancement in fluorescence in the presence of ATP and a small quench in the presence of ADP as a result of perturbing both the ground and excited state processes. Fluorescence lifetime and quantum yield measurements indicated that at least three microstates of Trp-501 were present in all nucleotide states examined, and these could not be assigned to a particular gross conformation of the motor domain. Enhancement in emission intensity was associated with a reduction of the contribution from a statically quenched component and an increase in a component with a 5-ns lifetime, with little change in the contribution from a 1-ns lifetime component. Anisotropy measurements indicated that the Trp-501 side chain was relatively immobile in all nucleotide states, and the fluorescence was effectively depolarized by rotation of the whole motor domain with a correlation time on 50-70 ns. Overall these data suggest that the backbone of the relay loop remains structured throughout the myosin ATPase cycle but that the Trp-501 side chain experiences a different weighting in local environments provided by surrounding residues as the adjacent converter domain rolls around the relay loop.
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PMID:The dynamics of the relay loop tryptophan residue in the Dictyostelium myosin motor domain and the origin of spectroscopic signals. 1127 75

Structural rearrangements of the myosin upper-50 kD subdomain are thought to play a key role in coordinating actin binding with nucleotide hydrolysis during the myosin ATPase cycle. Such rearrangements could open and close the active site in opposition to the actin-binding cleft, helping explain the opposing affinities of myosin for actin and nucleotide. To directly examine conformational changes across the active site during the ATPase cycle we have genetically engineered a mutant of chicken smooth-muscle myosin, F344W motor domain essential light chain, which contains a single tryptophan (344W) located on a short loop between two alpha helixes that traverse the upper-50 kD subdomain in front of the active site. Fluorescence resonance energy transfer was examined between the 344W donor probe and 2'(3')-O-(N-methylanthraniloyl) (mant)-nucleotide acceptor probes in the active site of this construct. The observed fluorescence resonance energy transfer efficiencies were 6.4% in the presence of mant ADP and 23.8% in the presence of mant ATP, corresponding to distances of 33.4 A and 24.9 A, respectively. Our results are consistent with structural rearrangements in which there is an 8.5-A closure between the 344W residue and the mant moiety during the transition from the strongly (ADP) to weakly (ATP) actin-bound states of the myosin ATPase cycle.
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PMID:Structural rearrangements in the active site of smooth-muscle myosin. 1595 90

Myosin is a motor protein associating with actin and ATP. It translates along actin filaments against a force by transduction of free energy liberated with ATP hydrolysis. Various myosin crystal structures define time points during ATPase showing the protein undergoes large conformation change during transduction over a cycle with approximately 10 ms periodicity. The protein conformation trajectory between two intermediates in the cycle is surmised by non-equilibrium Monte Carlo simulation utilizing free-energy minimization. The trajectory shows myosin transduction of free energy to mechanical work giving evidence for: (i) a causal relationship between product release and work production in the native isoform that is correctly disrupted in a chemically modified protein, (ii) the molecular basis of ATP-sensitive tryptophan fluorescence enhancement and acrylamide quenching, (iii) an actin-binding site peptide containing the free-energy barrier to ATPase product release defining the rate limiting step and, (iv) a scenario for actin-activation of myosin ATPase.
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PMID:Myosin dynamics on the millisecond time scale. 1791 31


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