Selectivity in substrate-enzyme complexation studied by surface forces measurement

The selectivity of substrate in substrate-enzyme complexation of heptaprenyl diphosphate synthase was directly investigated using colloidal probe atomic force microscopy (AFM). This enzyme is composed of two dissociable subunits, which exhibits a catalytic activity only when they are associated together in the presence of a cofactor, Mg²⁺, and a substrate, farnesyl diphosphate (FPP). We have recently succeeded to directly demonstrate a specific interaction involved in this enzyme reaction and obtain new insights into the molecular mechanism of the reaction using the approach based on the colloidal probe AFM. The AFM measurement showed the adhesive force between the subunits only in the presence of both Mg²⁺ and FPP. In this study, we studied the substrate selectivity in the complexation by monitoring the adhesive force. The substrates studied are pyrophosphate (PPi), isopentenyl diphosphate (IPP), geranyl diphosphate (GPP), farnesyl monophosphate (FP), and farnesyl geranyl diphosphate (FGPP). No adhesion was observed in the case of PPi, IPP, and GPP. On the other hand, the significant adhesion was observed for phosphate derivatives, which bear prenyl units longer than three. This is in good agreement with the selectivity of the substrates by this enzyme, which catalyzes the condensation reaction of four IPP molecules with FPP to give heptaprenyl (C₃₅) diphosphates. Our study showed a useful methodology for examining the elemental processes of biological reactions.