Allosteric activation unveils protein-mass modulation of ATP phosphoribosyltransferase product release

Heavy-isotope substitution into enzymes slows down bond vibrations and may alter transition-state barrier crossing probability if this is coupled to fast protein motions. ATP phosphoribosyltransferase from Acinetobacter baumannii is a multi-protein complex where the regulatory protein HisZ allosterically enhances catalysis by the catalytic protein HisG_S. This is accompanied by a shift in rate-limiting step from chemistry to product release. Here we report that isotope-labelling of HisG_S has no effect on the nonactivated reaction, which involves negative activation heat capacity, while HisZ-activated HisG_S catalytic rate decreases in a strictly mass-dependent fashion across five different HisG_S masses, at low temperatures. Surprisingly, the effect is not linked to the chemical step, but to fast motions governing product release in the activated enzyme. Disruption of a specific enzyme-product interaction abolishes the isotope effects. Results highlight how altered protein mass perturbs allosterically modulated thermal motions relevant to the catalytic cycle beyond the chemical step.