The nonessential nature of sulfhydryl groups for ATPase activity in myosin. A cyanylation study.

Cyanylation of myosin with 6-thiol’4Clcyanato-9-/3-o-ribofuranosylpurine shows a biphasic effect. During the incorporation of the first 4 mol of cyanide the Mg”+-dependent ATPase activity increases approximately &fold, the Ca*+ activity rises only slightly, and the K+(EDTA) activity falls to approximately 5% of its original value. These enzyme preparations can still be inactivated by N-ethylmaleimidel MgADP as is found for the native enzyme. Further modification (4 to 9.7 thiol groups) does not inactivate the enzyme as is the case with other thiol reagents. Instead, there is a sharp increase in Ca2+-ATPase activity together with a further increase in Mg’+ activity. No further change in K+ activity occurs, but a dramatic increase in protection against N-ethylmaleimide inactivatiou is observed. At maximal cyanylation (12.5 thiol groups) only 0.6 -SH group can be determined with Ellman’s reagent in a myosin preparation which was initially found to contain 39.2/malecule. After treatment of the cyanylated myosin with dithioerythritol almost all -SH groups can be detected again. This suggests that on maximal cyanylation, 26 -SH groups have formed disulfide bridges due to the cyanylation reagent. This is in agreement with the amount of reagent consumed during the cyanylation reaction and with the fact that addition of inorganic cyanide to the reaction mixture raises the number of incorporated cyanide groups to 261 molecule. The results show that cyanylated myosin retaining only 0.6 free sulfhydryl group is still active. This suggests that sulfhydryl groups are not directly involved in the mechanism of ATP hydrolysis, but that they affect the conformation of the active site.