In this report, we establish a straightforward method for estimating the equilibrium constant for the creatine kinase reaction (CK K_eq″) over wide but physiologically and experimentally relevant ranges of pH, Mg²⁺ and temperature. Our empirical formula for CK K_eq″ is based on experimental measurements. It can be used to estimate [ADP] when [ADP] is below the resolution of experimental measurements, a typical situation because [ADP] is on the order of micromolar concentrations in living cells and may be much lower in many in vitro experiments. Accurate prediction of [ADP] is essential for in vivo studies of cellular energetics and metabolism and for in vitro studies of ATP-dependent enzyme function under near-physiological conditions. With [ADP], we were able to obtain improved estimates of ΔG_ATP, necessitating the reinvestigation of previously reported ADP- and ΔG_ATP-dependent processes. Application to actomyosin force generation in muscle provides support for the hypothesis that, when [Pi] varies and pH is not altered, the maximum Ca²⁺-activated isometric force depends on ΔG_ATP in both living and permeabilized muscle preparations. Further analysis of the pH studies introduces a novel hypothesis around the role of submicromolar ADP in force generation.

In this report, we establish a straightforward method for estimating the equilibrium constant for the CK reaction over wide but physiologically and experimentally relevant ranges of pH, Mg2+ and temperature. Our empirical formula for CK Keq” is based on experimental measurements. It can be used to estimate [ADP] when [ADP] is below the resolution of experimental measurements, a typical situation because [ADP] is on the order of micromolar concentrations in living cells, and may be much lower in many in vitro experiments. Accurate prediction of [ADP] is essential for in vivo studies of cellular energetics and metabolism, and for in vitro studies of ATP-dependent enzyme function under near-physiological conditions. With [ADP], we could estimate ΔGATP. Application to actomyosin force generation in muscle provides support for the hypothesis that, when [Pi] varies but not when pH is altered, maximum Ca2+-activated isometric force depends on ΔGATP in both living and permeabilized muscle preparations. Further analysis of the pH studies introduces a novel hypothesis for the role of submicromolar ADP in force generation.