Nucleotide induced head-head interaction in myosin.

In isolated myosin the reaction sequence of essential thiol groups with N-ethylmaleimide was studied using the following five approaches: kinetics of the modification reaction, effects of modification on enzyme properties, affinity chromatography of isolated subfragment-1 stemming from modified myosin, isolation of cyanogen bromide peptides and identification of the tryptic thiol peptides thereof. All techniques involved revealed differences whether the modification was performed in the presence or absence of pyrophosphate on the one hand and in the presence of ADP or ATP on the other. In the former cases the two thiol-1 groups per myosin, one per active site, reacted at an equal rate indicating an equivalent microenvironment of these groups and hence a symmetric site-site relationship. In contrast, the nucleotides induce the sequential modification of thiol-1 on one head followed by the thiol-2 on the other head. This indicates non-equivalence in microenvironment of the essential thiols connected with each active site and hence that a form of asymmetric head-head interaction is operative.