| Abstract | The binding of the fluorescent analog of adenosine diphosphate (ADP)∗, 1,N6-ethenoadenosine diphosphate (εADP) to myosin and its subfragments, heavy meromyosin (HMM) and subfragment one (S1), has been studied under analagous conditions to those previously used in comparable studies on the binding of ADP to these molecules. The results indicate that there are two binding sites for εADP on myosin and HMM, and one site on S1. The dissociation constants for all had an identical value, within experimental error, of 2.0 (± .5) × 10−5 M−1. This is identical to the values found by Young (J. Biol. Chem., 242, 2790 (1967)) for ADP. In addition, the kinetics of hydrolysis of εATP versus ATP by S1 were studied. Values of Vmax and Km were 25 μM phosphate sec−1 (gm protein)−1 and 5 × 10−5 M−1 for ATP, and 80 μN phosphate sec−1 (gm protein)−1 and 45 × 10−5 M−1 for εATP. The results indicate that the increased Vmax that occurs when εATP is used as a substitute for ATP is not due to either an increased binding affinity of ATP for myosin and its subfragments, nor due to a decreased binding affinity of εATP versus ADP. This in turn suggests that the increase in Vmax may be due to an increased hydrolytic rate of εATP vs ATP in the enzyme substrate complex. |
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