The stability of biomolecules and the ability of proteins to perform their specific tasks is highly dependent on their physicochemical environment. In order to survive, living organisms maintain internal conditions that stabilize their underlying structures and allow the right biochemical reactions to take place. The ability of an enzyme to perform its task is influenced by the composition of its environment, like availability of certain ions and metals or presence of allosteric inhibitors or activators. To illustrate how our automated computer controlled framework can be used to investigate the influence of the physicochemical environment on enzyme activity, we NVP-BAG956 investigated the effects of KCl, pH and Fructose 1,6-bisphosphate on PYK activity while keeping substrate concentration constant at 1mM. We first measured the activity of PYK in 80 different mixtures that were chosen according to a space filling experimental design that covered the complete allowed concentration/pH range of all three variable components. Fig. 2 shows the measured data-points and two different slices of a Gaussian Random Process Regression model fitted to the data. It can be seen that all three variables strongly influence enzymatic activity. The optimum conditions determined from this model are KCl 90.6 mM, Fructose 1,6-bisphosphate 4.1 mM and pH 7.06. Enzymatic activity decreases quickly when deviating more than half a pH unit from the optimum pH either to the Pterostilbene acidic or basic side. Sufficiently high salt concentration is needed for the enzyme activity �C the activity drops quickly at KCl concentrations below 50 mM. High salt concentrations also inhibit the enzyme, but to a lesser extent. Fructose 1,6-bisphosphate is known as an allosteric activator that influences the KM of pyruvate kinase by changing its conformation when binding to it. Enzymatic activity at the substrate concentration used in this experiment decreases strongly if the Fructose 1,6-bisphosphate concentration is below 3 mM. Interestingly, high concentrations of Fructose 1,6-bisphosphate inhibit the enzyme as well. This behaviour has been reproduced in multiple similar experiments and, to our knowledge, has not been described in literature until now.