Interaction of ADP and Magnesium with the Active Site of Myosin Subfragment‐1 Observed by Reactivity Changes of the Critical Thiols and by Direct Binding Methods at Low and High Ionic Strength

Abstract

Comprehensive binding studies using direct and indirect methods yield stoichiometry and affinities for the binding of Mg · ADP and uncomplexed ADP to the active site of myosin subfragment‐1. Additionally, the binding parameters for Mg2+ in the ternary complex protein · Mg · ADP are presented for the first time. The indirect method makes use of reactivity changes of the critical thiol‐1 and thiol‐2 groups, which occur upon the binding of the ligand at the active site. The affinity constants derived by this method are corroborated by two independent direct methods, equilibrium dialysis and centrifugation transport. For Mg2+, ADP and Mg · ADP just one mole of ligand binds/mole subfragment‐1. The affinity of Mg · ADP at low ionic strength is 2.1 × 106 M‐1 and only five‐times lower in the absence of Mg2+ has a low affinity of 4.1 × 104 M‐1. At high ionic strength the uncomplexed ADP binds with a 43‐times‐lower affinity than Mg · ADP, whose affinity is 6.9 × 105 M−1. In this case Mg2+ interacts in the ternary complex with the higher affinity of 3.2 × 105 M−1, implying that at high salt concentration it plays a more prominent role in anchoring ADP at the active site.

The Role of Magnesium in Binding of the Nucleotide Polyphosphate Chain to the Active Site of Myosin Subfragment‐1

“…The inclusion of 5 mM EDTA in all solutions during protein preparation and storage, instead of thiol-protecting agents such as dithiothreitol or 2-mercaptoethanol, was found necessary to obtain consistent results of quantitative alkylation with radioactive MalNEt [7]. Both types of prcparations were identical, as characterized by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and their enzymic activities [4].…”

“…Equilibrium was reached after 20-35 h. After 60-72 h sample aliquots were drawn for determination of protein and Mg2+. Analysis of Mg2+ was carried out by atomic absorption (Perkin-Elmer 503 spectrometer) after deproteinization with suprapure perchloric acid [4].…”

“…Furthermore, reversible binding studies with ATP in the absence of divalent cations have led to the interpretation of a nonhydrolytic second site for ATP cxisting on myosin subfragment-I [3], which was postulated to have a regulatory function. We have recently shown that there is only one homogeneous binding site for the product ADP in both the presence and absence of divalent cations on subfragment-1 by employing direct and indirect methods [4]. It was also demonstrated that Mg2+ has only a relatively low affinity to the binary protein-ADP complex.…”

“…The more conventional direct methods, equilibrium dialysis and sedimentation, are no longer accurate enough for measuring affinities below lo4 M -' [4]. Therefore we included studies using quantitative affinity chromatography [6] and the indirect method based on ligand-induced changes in the reactivities of the critical thiol-1 and thiol-2 Enzyme.…”

“…Comparison with direct binding studies has demonstrated the success of this method in following the association of ADP with myosin subfragment-1 [4]. and metal standards for atomic absorption spectroscopy were from Merck (Darmstadt, FRG).…”