Analysis of positional isotope exchange in ATP by cleavage of the .beta.P-O.gamma.P bond. Demonstration of negligible positional isotope exchange by myosin

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Abstract
A method for analysis of positional isotope exchange (PIX) during ATP .dblarw. HOH oxygen exchange is presented that uses a two-step degradation of ATP resulting in cleavage of the .beta.P.sbd.O.gamma.P bond. This cleavage yields Pi derived from the .gamma.-phosphoryl of ATP that contains all four of the .gamma. oxygens. Both PIX between the .beta.,.gamma.-bridge and .beta.-nonbridge positions and washout of the .gamma.-nonbridge oxygens can be simultaneously followed by using ATP labeled with 17O at the .beta.-nonbridge positions and 18O at the .beta.,.gamma.-bridge and .gamma.-nonbridge positions. Application of this method to ATP .dblarw. HOH exchange during single turnovers of myosin indicates that the bulk of the ATP undergoes rapid washout of .gamma.-nonbridge oxygens in the virtual absence of PIX. At 25.degree. C with subfragment 1 the scrambling rate is at the limit of detectability of approximately 0.001 s-1, which is 50-fold slower than the steady-state rate. This corresponds to a probability of scrambling for the .beta.-oxygens of bound ADP of 1 in 10,000 for each cycle of reversible hydrolysis of bound ATP. A fraction of the ATP, however, does not undergo rapid washout. With myosin and stoichiometric ATP at 0.degree. C, this fraction corresponds to 10% of the ATP remaining at 36 s, or 2% of the initial ATP, and an equivalent level of ATP is found that does not bind irreversibly to myosin in a cold chase experiment. A significant level of apparent PIX is observed with fragment 1 in the fraction that resists washout, and this apparent PIX is shown to be due to contaminant adenylate kinase activity. This apparent PIX due to adenylate kinase provides a possible explanation for the PIX observed by Geeves et al. [Geeves, M. A., Webb, M. R., Midelfort, C. F., and Trentham, D. R. (1980) Biochemistry 19, 4748-4754] with subfragment 1.

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