Kinetic Investigation of the Ligand Dependence of Rabbit Skeletal Muscle Myosin Subfragment 1 Cys-697 and Cys-707 Reactivities

Publisher Website
Google Scholar
Abstract
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·MgATPγS > S1·MgADP > S1·MgPPi > S1·MgATP > actin·S1·MgADP > S1 > actin·S1 (for which ka ∼ 0 s-1). For PM, only ka can be measured for S1·MgATPγS 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·MgATPγS 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.