Realistic mathematical models of biochemical pathways require kinetic data that have been measured under physiological conditions. However, until recently most enzyme-kinetic data were obtained under conditions that yield maximum activity rather than physiological activity. The implementation of such non-physiological kinetic data is an important source of discrepancies between model simulations and corresponding experimental data. Here we review a number of initiatives to develop media for

more »

... me-kinetic assays that resemble the intracellular conditions of various organisms. We will discuss the challenges faced when developing such media, but also illustrate their relevance, since in vivo-like kinetic data improve the predictive value of kinetic models substantially.