The Time Dependence of the Activity of δ‐Chymotrypsin at High pH

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Abstract
When δ‐chymotrypsin at a high initial pH is mixed with a specific ester substrate, the hydrolytic reaction exhibits a lag phase that can be observed from the release of both products, the alcohol or the amino acid. This lag phase is under the dependence of the ionization of a group of pK 9, at 15°C, which has been previously shown to be the α‐amino group of isoleucine‐16. This lag corresponds to the time needed for the enzyme to isomerize from its “high‐pH” to its “neutral” conformation, and fluorescence measurements have been separately used to monitor this isomerization in the presence of substrate. The initial activity observed when the enzyme is mixed with the substrate is proportional to the concentration of enzyme present in its neutral conformation and is not significantly measurable when the enzyme is in its high‐pH conformation.It is concluded that the high‐pH form of δ‐chymotrypsin is inactive and cannot be involved in the observed activity of the enzyme at high pH. Therefore the activity of δ‐chymotrypsin exhibited at high pH is linked to a substrate‐promoted isomerization of the protein, that is, to a displacement of the conformational equilibrium presented by the protein towards its active neutral conformation.