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. This is accompanied by a shift in rate-limiting step from chemistry to product release. Here we report that isotope-labelling of HisG has no effect on the nonactivated reaction, which involves negative activation heat capacity, while HisZ-activated HisG catalytic rate decreases in a strictly mass-dependent fashion across five different HisG 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.