Effect of lysine methylation and other ATPase modulators on the active site of myosin subfragment 1.

Abstract

Many and diverse modifications of the myosin subfragment 1 (S-1) increase (modulate) its ATPase activity, including interaction of this particle with actin; a recent addition to these modifications is the extensive lysine modification of S-1 that seems prerequisite to crystallizing it for structure analysis. In this study we first established kinetically the ATPase modulations induced by various treatments of the myosin S-1 enzyme, and we also measured two properties of the S-1 active site-the affinity with which the site binds (a fluorescent analog of) the enzymatic nucleotide product and the access that a fluorescence quencher has to the bound ADP product-in an effort to get at the mechanism of modulation. Modulations achieved by substituting Ca2+ for the normal Mg2+ cocatalyst or by substituting Cl-for the normal carboxylate anion seem due to the product being held more loosely by the modulated enzyme. In other illustrative modulations (lysine methylation, or alkylation of or transition from neutral pH to pH 9.2) nucleotide product affinity and access to quencher do change, but not in a pattern explained simply by a lifting of product inhibition. Lysine methylation results in weaker binding of nucleotide product.Various modifications of myosin subfragment 1 (S-1) (including complexation with actin) modulate its ATPase rate, but the underlying mechanisms of modulation are unknown. This study looks for attendant active-site changes that may suggest a general mechanism. An immediate stimulus for this study was the S-1 modification reported most recently, the generalized lysine methylation (1) used to prepare myosin crystals for structural studies (2). White and Rayment (3) have reported that such methylation markedly increases the Mg2+-ATPase activity of S-1, in a manner kinetically similar to that produced by attaching a bulky group to 5). Do the seemingly disparate modulations such as those induced by generalized lysine methylation, modification of Cys-707, transition from pH 7.5 to 9.2, and many other agencies (6, 7) share a common feature with one another and perhaps with F-actin binding? Here we have studied three intentionally disparate modulations-methylation, alkylation of Cys-707, and the pH transition. Our efforts centered on two characterizations of the ATPase site, both deriving from an earlier observation (8) that, in the presence of an inoffensive concentration of the collisional fluorescence quencher acrylamide, a fluorescent nucleotide increased its emission upon binding to (being engulfed by) the ATPase site. By using this effect one can obtain the binding constant of the nucleotide to the site.Further, as Rosenfeld and Taylor (9) later showed, it is also possible to extract "Stern-Volmer quenching constants" of the nucleotide interacting with the site, thus measuring the access of the quencher to the bound nucleotide. These two measures-binding constant and Stern-Volmer constantexpress the physical state of the ATPase site and can be measured before ("control") and after modulating t...

Differential scanning calorimetric study of the complexes of modified myosin subfragment 1 with ADP and vanadate or beryllium fluoride

“…The Mg 2+-and Ca 2+-ATPase activities of $1 were enhanced by methylation sixfold and three-fold, respectively, while the K+-EDTAATPase activity was almost abolished (10-15% of the control). These changes in ATPase activities are similar to those described in the literature (Bivin et al, 1994;Phan et al, 1994).…”

Extensively Methylated Myosin Subfragment-1: Examination of Local Structure, Interactions with Nucleotides and Actin, and Ligand-Induced Conformational Changes

“…Our results on the release of trapped nucleotide from M-Sl and SI show that this aspect of coupling between actin and nucleotide sites is impaired in M-Sl. Despite the fact that the active site appears to be more "open" in M-Sl (Bivin et al, 1994) and releases trapped nucleotides at a faster rate than SI, the opposite holds in the presence of actin (Figure 3). The lack of actin movement over methylated HMM in the in vitro motility assay is consistent with the hypothesis that the tight coupling between the actin and nucleotide sites on SI and the structural changes underlying this coupling are essential for the motile function of myosin.…”

Tertiary Structural Changes in the Cleft Containing the ATP Sensitive Tryptophan and Reactive Thiol Are Consistent with Pivoting of the Myosin Heavy Chain at Gly699

“…If true, this characterization of the crystallographic structure might be attributed to the extensive methylation of lysine residues of S1 prior to crystallization (6) and/or to the temperature-dependent equilibrium between rigor-like and bound nucleotide-like conformations in S1 without nucleotide (53,54). In the former, the methylation of S1 is associated with altered ATPases and the abolishment of Trp510 sensitivity to the binding of ATP (55)(56)(57), both indicative of significant alteration of S1 structure particularly to the probe-binding cleft region. In the latter, higher temperatures favor the rigor-like conformation of S1 while lower temperatures, and perhaps the crystal, favor the nucleotide-like conformation.…”