The activity of triglyceride lipases is normaly investigated using a pH-stat and emulsified substrates. This method is relying on the release of protons concurrent with the enzymatic degradation of triglyceride lipids into diglycerides and charged free fatty acids at alkaline pH. The natural substrate for the triglyceride lipase is not the model emulsion, rather it is an insoluble lipidic phase consisting of multiple lipidic substances with no electrostatic charge.

Here we present an FT-IR attenuated total reflection report on cutinase that provides clear evidence for the enzymatic formation of neutral (protonated) fatty acids at pH 4. We propose a novel model for the observed acid activity. When the lipase attaches to the lipidic interphase, the active site is essentially covered with the lipid surface thus preventing chemical communication with the bulk solvent. Under these circumstances the classical pH definition may not hold due to the very limited volume combined with the low dielectric walls of the cavity.

At pH 4 the [H₃O⁺] concentration is 10^-4 - the average distance between two H₃O⁺ ions is approximately 200 Å. Assuming that the maximum dimension of the active site cavity is 8 Å, it is exceedingly unlikely that the His (H188) of the active site is protonated and that an H₃O⁺ simultaneously is present in the cavity. The protonation state of the active site His is thus not dictated by the solution pH but rather by the local environment, which is dictated by the protein component of the active site as well as the lipidic wall closing the entrance to the active site crevasse.