Intermediate oxygen exchange catalyzed by the actin-activated skeletal myosin adenosinetriphosphatase.
Considerable effort has been devoted to understanding the mechanism of 18O exchange in skinned skeletal and insect muscle fibers. However, a full understanding of the mechanism of 18O exchange in muscle fibers requires an understanding of the mechanism of 18O exchange in the simpler in vitro systems employing myosin subfragment 1 (S-1) and heavy meromyosin (HMM). In the present study, using both S-1 and S-1 covalently cross-linked to actin, we show first that over a wide range of temperature, ionic strength, and actin concentration there is only one pathway of 18O exchange with S-1. This is also the case with HMM except at very low ionic strength and low actin concentration, and even here, the data can be explained if 20% of the HMM is denatured in such a way that it shows no 18O exchange. Our results also show that actin markedly decreases the rate of 18O exchange. If it is assumed that Pi release is rate limiting, the four-state kinetic model of the actomyosin ATPase cannot fit these 18O exchange data. However, if it is assumed that the ATP hydrolysis step is rate limiting and Pi release is very fast, the four-state kinetic model can qualitatively fit these data although the fit is not perfect. A better fit to the 18O exchange data can be obtained with the six-state kinetic model of the actomyosin ATPase, but this fit requires the assumption that, at saturating actin concentration, the rate of Pi rotation is about 9-fold slower than the rate of reversal of the ATP hydrolysis step.
Evans
J A
JA
Laboratory of Cell Biology, National Heart, Lung, and Blood Institute, Bethesda, Maryland 20892.
Eisenberg
E
E
D000199:Actins / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D003432:Cross-Linking Reagents; D006868:Hydrolysis; D007527:Isoenzymes; D007700:Kinetics; D008956:Models, Chemical; D009132:Muscles / Q000201:enzymology; D015879:Myosin Subfragments / Q000378:metabolism; D009218:Myosins / Q000378:metabolism*; D009994:Osmolar Concentration; D010100:Oxygen / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism*; D013696:Temperature
D000199:Actins; D003432:Cross-Linking Reagents; D007527:Isoenzymes; D015879:Myosin Subfragments; D010446:Peptide Fragments; C033025:actin subfragments; D000255:Adenosine Triphosphate; D009218:Myosins; D010100:Oxygen