Kinetic investigation of the ligand dependence of rabbit skeletal muscle myosin subfragment 1 Cys-697 and Cys-707 reactivities.
Rate constants for the reactions of Cys-697 and Cys-707 of skeletal muscle myosin subfragment 1 (S1) with N,N'-p-phenylenedimaleimide (pPDM) and its monofunctional analog phenylmaleimide (PM) were measured for S1 and S1 bound to nucleotides and/or actin. The [pPDM] and [PM] dependencies indicate that prereaction noncovalent complexes of S1 and the alkylating agents form. The rates of the pseudo-first-order reactions of the complexes depend on the nucleotide bound. For pPDM, only the rate constant ka (for Cys-707 modification) can be measured. The relative ka magnitudes are S1. MgATPgammaS > S1.MgADP > S1.MgPPi > S1.MgATP > actin.S1.MgADP > S1 > actin.S1 (for which ka approximately 0 s-1). For PM, only ka can be measured for S1.MgATPgammaS and S1.MgPPi. However, for S1, S1. MgADP, and S1.MgATP, ki (for the reaction of Cys-697) can also be measured, and it is also nucleotide sensitive. The data are consistent with a mechanism in which pPDM or PM binds S1 near Cys-707 to form a noncovalent complex that reacts at a rate determined by the relative orientation of the cysteine sulfhydryl and the bound reagent. The simplest mechanism for the cross-linking step that reconciles these data with earlier cross-linker length data and with S1-nucleotide atomic structures is one which has pPDM-S1 complexes exist part of the time in conformations having the helical Cys-697/Cys-707-pPDM region converted to a loop structure which cross-links. The fact that rigor actin.S1 is the slowest and the S1.MgATP analog S1.MgATPgammaS is the fastest to be cross-linked is discussed in terms of possible energetic roles for helix to loop transitions of the Cys-697/Cys-707 region during the ATP hydrolysis cycle.
Polosukhina
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K
Department of Biochemistry, School of Dentistry, University of the Pacific, San Francisco, California 94115, USA.
Highsmith
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S
D000199:Actins / Q000378:metabolism; D000244:Adenosine Diphosphate / Q000378:metabolism; D000818:Animals; D003432:Cross-Linking Reagents / Q000494:pharmacology; D003545:Cysteine / Q000378:metabolism*; D007700:Kinetics; D008024:Ligands; D008301:Maleimides / Q000494:pharmacology; D018482:Muscle, Skeletal / Q000378:metabolism*; D015879:Myosin Subfragments / Q000378:metabolism*; D011487:Protein Conformation; D011817:Rabbits
D000199:Actins; D003432:Cross-Linking Reagents; D008024:Ligands; D008301:Maleimides; D015879:Myosin Subfragments; D000244:Adenosine Diphosphate; C011328:N,N'-4-phenylenedimaleimide; D003545:Cysteine