KINETIC CHARACTERIZATION OF MYOSIN HEAD FRAGMENTS WITH LONG-LIVED MYOSIN-CENTER-DOT-ATP STATES

We have separately expressed the Dictyostelium discoideum myosin II no nhydrolyzer point mutations E459V and E476K [Ruppel, K. M., and Spudic h, J., A. (1996) Mel. Biol. Cell 7, 1123-1136] in the soluble myosin h ead fragment M761-1R [Anson et al, (1996) EMBO J. 15, 6069-6074] and p erformed transient kinetic analyses to characterize the ATPase cycles of the mutant proteins. While the mutations cause some changes in mant ATP [2'(3')-O-(N-methylanthraniloyl)-ATP] and mantADP binding, the mos t dramatic effect is on the hydrolysis step of the ATPase cycle, which is reduced by 4 (E476K) and 6 (E459V) orders of magnitude. Thus, both mutant myosin constructs do in fact catalyze ATP hydrolysis but have very long-lived myosin ATP states. The E459V mutation allowed for a di rect measurement of the ATP off rate constant from myosin, which was f ound to be 2 x 10(-5) s(-1), Actin accelerated ATP release from this E 459V construct by at least 100-fold, Additionally, we found that the a ffinity of the E476K construct for actin is significantly weaker than for the wild-type construct, while the E459V mutant interacts with act in normally. Their functional properties and the fact that they can be produced and purified in large amounts make the E476K and E459V const ructs ideal tools to elucidate key structural features of the myosin A TPase cycle. These constructs should allow us to address important que stions, including how binding of ATP to myosin heads results in a >3 o rder of magnitude reduction in actin affinity.