Interactions of ATP Analogues, N⁶-[(6-Aminohexyl) Carbamoylmethyl] ATP and Its Dinitrophenyl Derivative, with the Active Site of Myosin

Abstract

The properties of N⁶-[(6-aminohexyl) carbamoylmethyl] ATP and its dinitrophenyl derivative as substrates of myosin were studied to test whether or not these ATP analogues are suitable for the affinity chromatography of myosin. The K_m and V_max values of heavy meromyosin for ATP, N⁶-[(6-aminohexyl)carbamoylmethyl]-ATP, and its dinitrophenyl derivative were 11.9 μM and 0.492 μmol P₁/min•mg, 137 μm and 0.454 μmol P₁min•mg, and 250 μm and 3.96 μmol P₁/min•mg, respectively in 0.5M KCI, 5mM CaCl₂, 20mM Tris-rnaleate buffer, pH 7.0 at 250°C. The association rate constant of N⁶-[6-aminohexyl) carbamoylmethyl] ATP to the active site of myosin was only 8% of that of ATP. The reason seemed to be the electrostatic repulsion between the positively charged group in the active site and the amino group of N⁶-[(6-aminohexyl) carbamoylmethyl]ATP because the rate constant of the dinitrophenyl derivative was 38% of that of ATP. It is suggested, therefore, that when this ATP analogue is attached to a solid matrix through its amino group, the association rate constant could be improved.
It was shown that the dinitrophenyl derivative of N⁶-[(6-aminohexyl) carbamoyl-methyl]ATP was trapped in the active site of heavy meromyosin by the cross-linking of SH₁ and SH₂ with p-phenylenedimaleimide. However, the stability of the dinitrophenyl derivative in the active site was very low compared to that of ATP. These results suggested that this ATP analogue interacts with myosin through the active site and that, if appropriate conditions to stabilize the complex with myosin are found, a column prepared with this analogue will be a useful tool for the affinity chromatography of myosin.
We suggest that the mechanism of nucleotide trapping is a conformational change in the active site which slows down the rate of product release rather than a steric blocking of the active site by the cross-linking reagent.