The combination of equilibrium measurements, microcalorimetry, and equilibrium modeling calculations allows for the characterization of the thermodynamic properties of biochemical reactions. The equilibrium and calorimetry measurements yield, respectively, values for apparent equilibrium constants K' and calorimetric molar enthalpies of reaction ΔrH(cal) for overall biochemical reactions. Overall biochemical reactions involve sums of species and this is a different situation than with the usually encountered reactions where only specific species participate in a reaction. Because of this, the values for K' and ΔrH(cal) vary with pH and pMg (pMg = ‑log10[Mg2+]) in addition to temperature T.
The dependence of K' on T, pH, and pMg can be calculated by using an equilibrium model. The equilibrium model can also be used to calculate values of the equilibrium constant K and standard molar enthalpy of reaction ΔrH° for a reference reaction that involves specific species. The aforementioned combination of tools has been applied to a large number of enzyme-catalyzed reactions, many of which are of significant interest to biotechnology and biomanufacturing. The methods will be described along with selected applications involving important biochemical reactions. The extant literature in this area has been examined and summarized in the form of several published reviews (J. Phys. Chem. Ref. Data 2007, 36, 1347–1397 and references cited therein) and is also available as an online database: https://randr.nist.gov/enzyme/Default.aspx.