Many enzymes catalyze the abstraction of a proton from a carbon acid substrate to initiate a variety of reactions; however, the development of a complete quantitative description of enzyme-catalyzed heterolytic cleavage of a C–H bond remains a challenge to enzymologists. To determine the pK^C–H_a value for such substrates bound at the active site, recent studies have estimated the equilibrium for formation of the deprotonated intermediate at the active site, however, accurate knowledge of the pK^BH+_a of the conjugate acid of the Brønsted base catalyst (BH⁺) is also required. Herein, it is shown that using the value of pK^BH+_a of the enzyme–substrate complex can underestimate the value of pK^C–H_a by an amount between zero and pδ, where pδ is the change in basicity of BH⁺ upon going from the enzyme–substrate complex to the enzyme-intermediate complex.