Oxygen-exchange studies on the pathways for magnesium adenosine 5'-triphosphate hydrolysis by actomyosin

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Abstract
At an intermediate stage in the hydrolysis of MgATP by [rabbit muscle] myosin or actomyosin, there is an exchange of O2 between water and the P.gamma. group of enzyme-bound nucleotide. Starting with [P.gamma.-18O]ATP as substrate, the exchange is revealed in the [18O]Pi species that are ultimately released as product into the reaction medium. An analysis of the distribution of these labeled Pi species, which contain 3, 2, 1 or none of the 18O atoms originally on the P.gamma. of ATP, is used to probe intermediate stages of the hydrolytic mechanism. More than 1 pathway of hydrolysis operates. Two of these pathways are spurious; 1 is a nonexchanging MgATPase that is present in fresh myosin preparations; the other is an induced slow exchange that develops in myosin during storage (-20.degree. C) and subsequent aging (4.degree. C). After correction for these artifacts, 2 normal pathways for actomyosin hydrolysis remain. These normal pathways differ in the mode of interaction between actin and myosin in the course of hydrolysis; one is the Lymn-Taylor pathway where O2 exchange occurs at a stage when actin and myosin are dissociated; the other is a pathway in which actin and myosin are associated during O2 exchange. Each of these 2 pathways contributes an equal amount of Pi to the product pool. Thus, on average, each myosin head uses each of these pathways half the time. Evidently, during contraction, myosin can dissociate from the actin filament only during every other cycle of MgATP hydrolysis or that only half the heads, at any one time, can exchange O2 while free of the actin filament.
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