Biological systems are acknowledged to be robust to perturbations but a rigorous understanding of this has been elusive. In a mathematical model, perturbations often exert their effect through parameters, so sizes and shapes of parametric regions offer an integrated global estimate of robustness. Here, we explore this “parameter geography” for bistability in post-translational modification (PTM) systems. We use the previously developed “linear framework” for timescale separation to describe the steady-states of a two-site PTM system as the solutions of two polynomial equations in two variables, with eight non-dimensional parameters. Importantly, this approach allows us to accommodate enzyme mechanisms of arbitrary complexity beyond the conventional Michaelis-Menten scheme, which unrealistically forbids product rebinding. We further use the numerical algebraic geometry tools Bertini, Paramotopy, and alphaCertified to statistically assess the solutions to these equations at ∼109 parameter points in total. Subject to sampling limitations, we find no bistability when substrate amount is below a threshold relative to enzyme amounts. As substrate increases, the bistable region acquires 8-dimensional volume which increases in an apparently monotonic and sigmoidal manner towards saturation. The region remains connected but not convex, albeit with a high visibility ratio. Surprisingly, the saturating bistable region occupies a much smaller proportion of the sampling domain under mechanistic assumptions more realistic than the Michaelis-Menten scheme. We find that bistability is compromised by product rebinding and that unrealistic assumptions on enzyme mechanisms have obscured its parametric rarity. The apparent monotonic increase in volume of the bistable region remains perplexing because the region itself does not grow monotonically: parameter points can move back and forth between monostability and bistability. We suggest mathematical conjectures and questions arising from these findings. Advances in theory and software now permit insights into parameter geography to be uncovered by high-dimensional, data-centric analysis.

Robustness and parameter geography in post-translational modification systems