The role of magnesium in binding of the nucleotide polyphosphate chain to the active site of myosin subfragment-1.
The binding of adenosine 5'-[beta, gamma-imido]triphosphate, pyrophosphate and triphosphate to the active site of myosin subfragment-1 was assessed in the presence and absence of Mg2+ by direct and indirect methods. In addition, the affinity and stoichiometry of Mg2+ in the ternary complexes formed by protein, Mg2+ and each of these phosphate compounds have been determined. As direct methods, equilibrium dialysis, sedimentation and quantitative affinity chromatography were used in conjunction with the indirect method of monitoring reactivity changes of the critical thiol-1 and thiol-2 groups, which occur upon binding of the ligands at the active site. There was good agreement between the results yielded by the different methods. All three phosphate compounds alone bind just one molecule per isolated myosin head portion with similar affinities lying in the range 1-4 X 10(3) M-1. Again only one molecule/head portion binds when they exist in the form complexed with Mg2+, but now show much higher affinities of between 10(6)-10(7) M-1. In all cases Mg2+ was found to be associated in the ternary complexes with the very high affinity of 10(8)-10(9) M-1. It is postulated that this ion plays a prominent role in fixing the phosphate chain in the myosin active site. In contrast, Mg2+ scarcely affects the affinity of ADP and shows only a low affinity around 4 X 10(4) M-1 in the ternary complex [Watterson, J.G., Foletta, D., Kunz, P.A., and Schaub, M. C. (1983) Eur. J. Biochem. 131, 89-96]. As pyrophosphate displays binding parameters similar to the triphosphate compounds and widely different from ADP, it is argued that it may bind in the beta, gamma-phosphate positions at the active site.
D000227:Adenine Nucleotides / Q000378:metabolism*; D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000266:Adenylyl Imidodiphosphate / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002846:Chromatography, Affinity; D003956:Dialysis; D008274:Magnesium / Q000378:metabolism*; D015879:Myosin Subfragments; D009218:Myosins / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism*; D011817:Rabbits
D000227:Adenine Nucleotides; D015879:Myosin Subfragments; D010446:Peptide Fragments; D000266:Adenylyl Imidodiphosphate; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D009218:Myosins; D008274:Magnesium