Amphoteric charge distribution at the enzymatic site of 1,N6-ethenoadenosine triphosphate-binding heavy meromyosin determined by dynamic fluorescence quenching.
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 (epsilon-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 TI+ 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 TI+ and I- for epsilon-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 ad both TI+ 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 epsilon-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 epsilon-adenine ring and a negative charge is located on the opposite side. The negative charge is attributed to the phosphate group in epsilon-ATP and positive one is probably attributable to a lysyl residue in the polypeptide chain of HMM.
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives*; D000818:Animals; D001665:Binding Sites; D004985:Ethenoadenosine Triphosphate / Q000494:pharmacology*; D007700:Kinetics; D009132:Muscles / Q000201:enzymology; D015879:Myosin Subfragments / Q000378:metabolism*; D009994:Osmolar Concentration; D011485:Protein Binding; D011817:Rabbits; D013050:Spectrometry, Fluorescence
D004985:Ethenoadenosine Triphosphate; D015879:Myosin Subfragments; D000255:Adenosine Triphosphate