MAGNESIUM-ION DEPENDENT RABBIT SKELETAL-MUSCLE MYOSIN GUANOSINE AND THIOGUANOSINE TRIPHOSPHATASE MECHANISM AND A NOVEL GUANOSINE DIPHOSPHATASE REACTION

The mechanism of the Mg2+-dependent myosin subfragment 1 catalyzed hydrolysis of GTP and 2-amino-6-mercapto-9-β-ribofuranosylpurine 5‘-triphosphate (thioGTP) has been investigated by rapid-reaction techniques. The myosin was isolated from rabbit skeletal muscle. The steady-state intermediate of these reactions consists predominantly of a protein-substrate complex unlike the myosin subfragment 1 ATPase reaction which has a protein-products complex as the principal steady-state component. The mechanism of GTP hydrolysis catalyzed by subfragment 1 has other marked differences from the ATPase mechanism. The second-order rate constant of binding of GTP to subfragment 1 is tenfold greater than that for GDP binding. The dissociation rate constant of GDP from subfragment 1 is 0.06 s-1 compared with the subfragment 1 catalytic center activity for GTP hydrolysis of 0.5 s-1 at pH 8.0 and 20 °C. This shows that GDP bound to subfragment 1 forms a complex which is not kinetically competent to be an intermediate of the GTPase mechanism. GDP is hydrolyzed in the presence of subfragment 1 to GMP and P1. The subfragment 1 GTPase mechanism has a number of features in common with that of the elongation factor Tu GTPase of the protein biosynthetic system of Escherichia coli. © 1979, American Chemical Society. All rights reserved.