Kinetics of nucleoside triphosphate cleavage and phosphate release steps by associated rabbit skeletal actomyosin, measured using a novel fluorescent probe for phosphate

We have measured the kinetics of inorganic phosphate (P-i) release during a single turnover of actomyosin nucleoside triphosphate (NTP) hydrolysis using a double-mixing stopped-flow spectrofluorometer, at very low ionic strength to increase the affinity of myosin-ATP and myosin-ADP-P-i to actin, Myosin subfragment I and a series of nucleoside triphosphates were mixed and incubated for similar to 1-10 s to allow NTP to bind to myosin and generate a steady state mixture of myosin-NTP and myosin-NDP-P-i. The steady state intermediates were then mixed with actin. The kinetics of P-i release were measured using a fluorescent probe for P-i, based on a phosphate binding protein [Brune et al. (1994) Biochemistry 33, 8262-8271], These data are correlated with quenched-flow data, where the extent of the rapid burst of hydrolysis during the first turnover of ATF hydrolysis was followed by chemical quenching of the reaction mix al Various times after rapidly mixing ATP and myosin subfragment 1. From the double-mixing actomyosin measurements, the kinetics of P-i release are biphasic. The fast phase corresponds to P-i release from the associated actomyosin-ADP-P-i complex. The slow phase measures the rate of the cleavage step on associated actomyosin, At saturating actin, there is a correlation between the amplitude of the fast phase and the size of the P-i burst observed by quenched flow in the absence of actin: the size of this phase corresponds to the amount of myosin-ADP-P-i formed during the first mix, For ATP at 20 degrees C the rate of the P-i release step is 75 (+/-5) s(-1), 25-fold larger than the cleavage step, which is the rate-limiting step of actomyosin ATP hydrolysis at saturating actin. The rate constant of P-i release varies only slightly with nucleoside structure, The rate constant of the slow phase of the P-i release (measuring cleavage) is highly dependent upon the structure of the NTP substrate.