Amphoteric Charge Distribution at the Enzymatic Site of 1,N6-Ethenoadenosine Triphosphate-Binding Heavy Meromyosin Determined by Dynamic Fluorescence Quenching1

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Abstract
The features of the charge distribution in the vicinity of the ATP-binding site of heavy meromyosin (HMM) were investigated by the technique of dynamic fluorescence quenching. Instead of ATP, 1,N6-ethenoadenosine triphosphate (E-ATP), a fluorescent derivative of ATP, was attached to the ATP-binding site in the presence of an ATP-regenerating system. The I- ion and acrylamide were used as negative-and zero-charged quenchers. In addition to these quenchers, we used the Tl+ ion, which has recently been found to be a highly efficient quencher with positive charge, and to be generally applicable to fluorescence-labeled proteins. The Stern-Volmer quenching constants of Tl+ and I− for ɛ-ATP bound to HMM both decreased with increasing ionic strength of the solvent. This result means that there is an electrostatic attractive force between the fluorophore and both Tl+ and I−. On the other hand, the Stern-Volmer quenching constant of acrylamide was not significantly affected by a change in ionic strength. This result confirms that no significant change in protein conformation in the vicinity of the ɛ-ATP-binding site of HMM occurs with change in ionic strength. In order to interpret these results, we propose a model in which a positive charge is located on one side of the ɛ-adenine ring and a negative charge is located on the opposite side. The negative charge is attributed to the phosphate group in ɛ-ATP and the positive one is probably attributable to a lysyl residue in the polypeptide chain of HMM.