The active site of myosin adenosine triphosphatase. I. Localization of one of the sulfhydryl groups.

It is now widely recognized that under certain conditions of temperature and pH (1, 2)) the Ca++-activated hydrolysis of adenosine triphosphate (ATP) by myosin exhibits a biphasic response to titration of the sulfhydryl groups of the protein by organic mercurials and by N-ethylmaleimide (3-5). On the other hand, titration by these reagents results only in inhibition if inosine triphosphate or guanosine triphosphate are the substrates or if ethylenediaminetetraacetate (EDTA) is used as the activator with ATP (3-6). It has become clear, also, that this protein is built up from physically and chemically identical subunits or monomers with a molecular weight of approximately 200,000 (7-9). Although participation of some low molecular weight component in the structure is not excluded, it appears that the basic form of the molecule is a three-stranded rope built up from these highly helical subunits (7). With the development of the radioactive labeling and “fingerprint” technique employed in demonstrating that the subunits are probably of identical chemical structure (9)) it became possible to approach the problem of the structural segments involved in these sulfhydryl-binding effects. Early realization that the reaction of N-ethylmaleimide with myosin -SH was quite selective in inhibition of the EDTA-activated adenosine triphosphatase (3) encouraged us to utilize partial labeling with W-N-ethylmaleimide in an effort to locate that portion of the structure containing the cysteine residue or residues involved in loss of the EDTA activation, with the hope that eventually the structure of the active site or sites might be identified. The present report demonstrates that the sensitivity of the EDTAactivated adenosine triphosphatase is associated with the masking of one of two SH groups present in one of the peptides resulting from exhaustive trypsin digestion of myosin.