| Abstract | The fluorescent analog of adenosine triphosphate (ATP)∗ 1,N6-ethenoadenosine triphosphate, (εATP), has been utilized as a substitute for ATP in the myosin and heavy meromyosin ATPase systems. For myosin, the analog εATP replaced ATP with a somewhat larger Km (2.6 × 10−4 mole ℓ−1 for εATP as opposed to 8.8 × 10−5 mole ℓ−1 for ATP), indicating that the apparent affinity of the enzyme for εATP is less than for ATP. Perhaps of more interest, further comparison yielded a Vmax for εATP about two and one half times the value for ATP (20 μmole PO4 sec−1 g protein−1 as opposed to 8.1 μmole sec−1 g protein−1). Results for the HMM-εATPase system were similar, yielding a Km value of 1.47 × 10−4 mole ℓ−1 and a Vmax of 54.2 μmole PO4 sec−1 g protein−1, as opposed to corresponding Km and Vmax values of 1.23 × 10−4 mole ℓ−1 and 20.4 μmole PO4 sec−1 g protein−1, respectively for the HMM-ATP interaction. The pH dependence of εATPase for both systems was comparable to ATP, suggesting a similarity in the mechanism of hydrolysis of the two nucleotides. Activation of εATPase by Ca2+ in the presence of 0.5 M KCl was comparable to ATPase for both systems, but inhibition by Mg2+ seemed to be more effective for εATPase. These results indicate that εATP is an excellent substitute for ATP in the myosin and heavy meromyosin systems and because of its insertion into the active site of these muscle proteins, it promises to be a very useful probe for conformation studies at this level. |
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