Transient kinetics of ATP hydrolysis by covalently crosslinked actomyosin complex in water and 40% ethylene glycol by the rapid flow quench method

Publisher Website
Google Scholar
Abstract
The initial steps of the ATPase of covalently cross-linked actomyosin subfragment 1 (acto-SF-1) were studied by the rapid flow quench method, and the results obtained were compared with those with reversible (i.e., non-cross-linked) acto-SF-1 and SF-1 under identical conditions. Cross-linked acto-SF-1 plus [.gamma.-32P]ATP reaction mixture milliseconds old was quenched either in a large excess of unlabeled ATP (ATP chase) or in acid (Pi burst). The conditions were pH 8 and 15.degree. C at 5 mM or 0.15 M KCl and with or without 40% ethylene glycol. In 40% ethylene glycol (5 mM KCl), as with SF-1 and reversible acto-SF-1, the ATP chase was used to titrate active sites and to study the kinetics of ATP binding. Unlike those with SF-1 or reversible acto-SF-1, saturation kinetics were not obtained. The 2nd-order rate constant for ATP binding ws 3.1 .times. 106 M-1 s-1 for cross-linked acto-SF-1, 1.8 .times. 106 M-1 s-1 for reversible acto-SF-1, and 2 .times. 106 M-1 s-1 for SF-1. In Pi burst experiments, a transient phase could not be discerned. Because of a high kcat, cross-linked acto-SF-1 was difficult to study in aqueous solution, but at 5 mM KCl, the ATP chase and Pi burst curves were similar to those obtained in 40% ethylene glycol. At 0.15 M KCl the ATP chase curve was difficult to interpret (small amplitude), and there was a small Pi burst. The data obtained in 40% ethylene glycol (5 mM KCl) were adjusted to an abbreviated form of the Bagshaw-Trentham scheme for myosin: .**GRAPHIC**. The best fit was obtained with K1k2 = 3.1 .times. 106 M-1 s-1 (K1 < 1.06 .times. 104 M-1, k2 > 200 s-1), .kappa.3 = k-3 = 4 s-1 and k4 = 20 s-1. With SF-1, K1 = 1.25 .times. 105 M-1, k2 = 16 s-1, k3 = k-3 = 8 s-1 and k4 = 0.12 s-1 under the same conditions. Whereas the kinetics of the ATP binding and the release of products for cross-linked acto-SF-1 and SF-1 appear very different, the kinetics of their chemical steps seem similar.
Keywords