Synthesis of non-nucleotide ATP analogues and characterization of their chemomechanical interaction with muscle fibres

To probe the substrate requirements for the actomyosin chemomechanical interaction, the effects of a series of eight new non-nucleotide ATP analogues on actomyosin-catalysed hydrolysis rates and on fibre mechanics have been investigated. These analogues have substitutions of new functional groups at the 2- and 4- positions of the ATP analogues, 2-[(4-azido-2-nitrophenyl)amino]ethyl triphosphate (NANTP), and 3-[(4-nitrophenyl)amino]propyl triphosphate (PrNANTP). Previous work has shown NANTP but not PrNANTP will support active tension and shortening in skinned muscle fibres in a manner almost identical to ATP. Here all 2- and 4- phenyl substituted analogues had myosin subfragment 1 (S1) NTPase hydrolysis rates higher than ATP and the rates were stimulated by addition of actin. In general, the replacement of the 4-azido group of NANTP with-H,-NO2 or-NH2 had small effects on fibre mechanics while replacement of 2-NO2 group with-H or-NH2 dramatically lowered the ability of the new analogues to support active tension and shortening. All PrNANTP-based analogues were ineffective in supporting active tension or shortening. We found no correlation between S1 or actoS1 NTPase rates and any mechanical parameters. However, for all analogues there was a strong correlation between the maximal velocity of shortening (Vmax) and isometric tension (Po). A three-state, chemomechanical model is proposed in which the analogues effect the transition rate into a strongly-bound, force-producing crossbridge state to account for this correlation. These studies identify 2-[(2-nitrophenyl)amino]ethyl triposphate as the chemically simplest ATP analogue which closely mimics the effect of ATP in skinned muscle fibres.