Mechanism for coupling free energy in ATPase to the myosin active site

Sungjo Park, Katalin Ajtai, Thomas P Burghardt

Research output: Contribution to journalArticle

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Abstract

Acrylamide quenching of tryptophan 510 (Trp510) fluorescence in rabbit skeletal myosin subfragment 1 (S1) indicates the conformation of the probe binding cleft, containing the highly reactive thiol (SH1) and Trp510, in the presence of nucleotides or nucleotide analogs trapped in the active site of S1 [Park et al. (1996) Biochim. Biophys. Acta 1296, 1-4]. The Trp510 quenching efficiency shows that the probe binding cleft closes slightly in the presence of beryllium fluoride trapped MgADP (MgADPBcF, -S1) and most tightly in the presence of vanadate trapped MgADP (MgADPVi-S1) with aluminum fluoride and scandium fluoride trapped MgADP (MgADPAIF4-S1 and MgADPScF(x)- S1) falling in between in the order MgADPBeF(x) > MgADPAIF4 > MgADPScF3 > MgADPVi. These nucleotide analogs are identified with sequential structural changes in MgATP during hydrolysis in the same order with beryllium fluoride occurring earliest in the ATPase cycle. Correlation of the separation distance of the γ-phosphate analog metal from the oxygen connecting it to the β-phosphate in ADP, to the extent of cleft closure, suggests that this distance in the nucleotide transition state determines the conformation of the probe binding cleft. Trp510 quenching efficiency was also measured as a function of the base moiety of the vanadate trapped Mg-nucleotide diphosphate (MgNDPVi-S1). The extent of cleft closure is largest in the presence of the natural substrate NDP and follows the order MgADPVi > MgCDPVi > MgUDPVi > MgIDPVi > MgGDPVi with very little difference between MgADPVi and MgCDPVi. These data follow the order of the effectiveness of the corresponding nucleotide triphosphates to support force production in muscle fibers [Pate et al. (1993) J. Biol. Chem. 268, 10046-10053]. In both the fiber and S1, it appears that the 6-position amino group of the bases of ADP and CDP is required to properly anchor the nucleotide in the active site, possibly at tyrosine 135 as suggested by X ray crystallographic studies [Fisher et al. (1995) Biochemistry 34, 8960-8972]. Finally, the Trp510 quenching efficiency was measured as a function of the size of the divalent cation trapped in the active site of S1 with ADPVi. These data failed to show a correlation suggesting that the divalent cation is not involved with the propagation of influence from the active site to the probe binding cleft. The forgoing experiments suggest that the changing conformation of ATP during hydrolysis, parameterized by the increasing distance between the β- and the γ-phosphate, stresses the active site of S1 through protein- nucleotide contacts at the γ-phosphate and nucleotide base. The stress induced strain in the cross-bridge may be the mechanism by which energy in ATP is transferred to the myosin structure.

Original languageEnglish (US)
Pages (from-to)3368-3372
Number of pages5
JournalBiochemistry
Volume36
Issue number11
DOIs
StatePublished - Mar 18 1997

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Myosins
Free energy
Adenosine Triphosphatases
Catalytic Domain
Nucleotides
Tryptophan
Adenosine Diphosphate
Quenching
Phosphates
Conformations
Vanadates
Adenosine Triphosphate
Divalent Cations
Hydrolysis
Scandium
Cytidine Diphosphate
Myosin Subfragments
Biochemistry
Acrylamide
Fibers

ASJC Scopus subject areas

  • Biochemistry

Cite this

Mechanism for coupling free energy in ATPase to the myosin active site. / Park, Sungjo; Ajtai, Katalin; Burghardt, Thomas P.

In: Biochemistry, Vol. 36, No. 11, 18.03.1997, p. 3368-3372.

Research output: Contribution to journalArticle

Park, S, Ajtai, K & Burghardt, TP 1997, 'Mechanism for coupling free energy in ATPase to the myosin active site', Biochemistry, vol. 36, no. 11, pp. 3368-3372. https://doi.org/10.1021/bi9624999
Park, Sungjo ; Ajtai, Katalin ; Burghardt, Thomas P. / Mechanism for coupling free energy in ATPase to the myosin active site. In: Biochemistry. 1997 ; Vol. 36, No. 11. pp. 3368-3372.
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N2 - Acrylamide quenching of tryptophan 510 (Trp510) fluorescence in rabbit skeletal myosin subfragment 1 (S1) indicates the conformation of the probe binding cleft, containing the highly reactive thiol (SH1) and Trp510, in the presence of nucleotides or nucleotide analogs trapped in the active site of S1 [Park et al. (1996) Biochim. Biophys. Acta 1296, 1-4]. The Trp510 quenching efficiency shows that the probe binding cleft closes slightly in the presence of beryllium fluoride trapped MgADP (MgADPBcF, -S1) and most tightly in the presence of vanadate trapped MgADP (MgADPVi-S1) with aluminum fluoride and scandium fluoride trapped MgADP (MgADPAIF4-S1 and MgADPScF(x)- S1) falling in between in the order MgADPBeF(x) > MgADPAIF4 > MgADPScF3 > MgADPVi. These nucleotide analogs are identified with sequential structural changes in MgATP during hydrolysis in the same order with beryllium fluoride occurring earliest in the ATPase cycle. Correlation of the separation distance of the γ-phosphate analog metal from the oxygen connecting it to the β-phosphate in ADP, to the extent of cleft closure, suggests that this distance in the nucleotide transition state determines the conformation of the probe binding cleft. Trp510 quenching efficiency was also measured as a function of the base moiety of the vanadate trapped Mg-nucleotide diphosphate (MgNDPVi-S1). The extent of cleft closure is largest in the presence of the natural substrate NDP and follows the order MgADPVi > MgCDPVi > MgUDPVi > MgIDPVi > MgGDPVi with very little difference between MgADPVi and MgCDPVi. These data follow the order of the effectiveness of the corresponding nucleotide triphosphates to support force production in muscle fibers [Pate et al. (1993) J. Biol. Chem. 268, 10046-10053]. In both the fiber and S1, it appears that the 6-position amino group of the bases of ADP and CDP is required to properly anchor the nucleotide in the active site, possibly at tyrosine 135 as suggested by X ray crystallographic studies [Fisher et al. (1995) Biochemistry 34, 8960-8972]. Finally, the Trp510 quenching efficiency was measured as a function of the size of the divalent cation trapped in the active site of S1 with ADPVi. These data failed to show a correlation suggesting that the divalent cation is not involved with the propagation of influence from the active site to the probe binding cleft. The forgoing experiments suggest that the changing conformation of ATP during hydrolysis, parameterized by the increasing distance between the β- and the γ-phosphate, stresses the active site of S1 through protein- nucleotide contacts at the γ-phosphate and nucleotide base. The stress induced strain in the cross-bridge may be the mechanism by which energy in ATP is transferred to the myosin structure.

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