Scalable biophysical constraints for physiologically consistent metabolic states

Systems biology aims to develop predictive models that connect molecular mechanisms to cellular behavior. Genome-scale metabolic models are among the most widely used frameworks for integrating stoichiometric, thermodynamic, and omics-derived information to predict feasible metabolic phenotypes. However, cellular metabolism operates on timescales governed by enzyme kinetics and by the relationship between metabolic fluxes and metabolite pool sizes. In steady-state metabolic models, this relationship can be expressed in terms of metabolite turnover rates, defined as flux-to-pool-size ratios that quantify how rapidly metabolite pools are renewed. As a result, physiologically consistent steady-state solutions should not only satisfy mass-balance and thermodynamic constraints but also exhibit turnover rates consistent with enzyme-mediated cellular dynamics. Current constraint-based approaches can admit many steady-state flux-concentration states that do not account for turnover rates, resulting in phenotypes incompatible with realistic metabolic dynamics, even when multiple types of data are imposed. Here, we present METEOR-K, an optimization framework that links steady-state metabolic fluxes to metabolite concentrations via turnover rate constraints to identify dynamically plausible flux-concentration reference states. Because these constraints reshape the feasible solution space, we also introduce turnover-rate-aware sampling strategies to efficiently explore the resulting feasible region. We applied METEOR-K to models of increasing scope and scale, including a reduced glycolysis pathway, anaerobic E. coli, and near-genome-scale ovarian cancer models. METEOR-K narrowed the admissible steady-state solution space, reduced uncertainty in feasible flux-concentration states, and improved local dynamic behavior. In nonlinear ODE simulations of bioreactor cultivation and drug-response scenarios, METEOR-K-derived states produced intracellular response times compatible with growth-supporting metabolic operation and perturbation recovery. Overall, these results establish metabolite turnover rates as scalable biophysical constraints that improve the physiological consistency of steady-state metabolic modeling. Because turnover rates encode flux-to-pool-size timescale constraints, METEOR-K moves part of physiological-consistency assessment upstream of kinetic parameterization, yielding better-suited flux-concentration reference states for kinetic modeling and dynamic prediction.