The Active Site of Myosin A-Adenosine Triphosphatase*

Abstract

Myosin was treated with DHT (diazonium-1H-tetrazole) in the presence or absence of Mg-ATP. Results indicated that the SH-groups of myosin were modified by DHT. To prevent reaction of the SH-groups of myosin with DHT, the groups were blocked reversibly by DGM through their conversion to disulfide bonds. The Ca2+.ATPase [EC 3.6.1.3] activity of myosin disappeared on treatment with DGM (dithiodiglycolic acid dimethyl ester) for 50 hr and the amount of free SH-groups decreased to 2 moles per mole of myosin. The Ca2+ATPase activity was restored to the original level by reversing the blocking reaction with excess βME or DTT. DGM-treated myosin was treated with DHT, and then with β-ME or DTT. The Ca2+-ATPase activity decreased linearly with increase in the amount of monoazotyrosine in myosin, and complete inactivation occurred when approximately one mole of monoazo-tyrosine was formed per 4.0×105g of myosin. During treatment of DGM-treated myosin with DHT, the Ca2+-ATPase activity was protected to some extent by the presence of Mg-ATP. H-Meromyosin prepared by tryptic digestion of azo-myosin had the same monoazo-tyrosine content as the parent myosin. All the monoazo-tyrosine was present in the f-subunit. When a few moles of tyrosine residues per mole of myosin were modified with DHT, myosin became soluble even in water. One mole of tyrosine residue per mole of L-meromyosin fraction 1 reacted specifieally with DHT, and filament formation in L-meromyosin fraction 1 was inhibited by this modification with DHT.