The Conformational Oscillation of δ‐Chymotrypsin Involvement of Methionine‐192

In δ‐chymotrypsin the reactivity of methionine‐192 towards p‐nitrophenacyl bromide is strongly reduced when the α‐amino group of isoleucine‐16 has been acetylated. Since acetylation of isoleucine‐16 brings δ‐chymotrypsin to a conformation similar to its alkaline one this suggests that methionine‐192 should present an impaired reactivity in the alkaline conformation of the protein. It is indeed observed that its chemical reactivity as a function of pH depends on the ionization state of the α‐amino group of isoleucine‐16 (pKapp 9 at 15 °C), as does the structure of the enzyme. Reciprocally, after chemical reaction of methionine‐192 with hydrogen peroxide, isoleucine‐16 presents a slower rate of reaction with fluorescamine than when methionine‐192 is free. As a result of methionine‐192 oxidation the apparent pK of the alkaline transition is shifted from 9 to about 11 at 15 °C. This is reflected in the disappearance of the lag phase previously observed for the initial activity of the enzyme when it is incubated at alkaline pH [Eur. J. Biochem. (1973) 39, 293 – 300]. The absence of chemical reactivity of methionine‐192 in the alkaline state of the enzyme is confirmed by the appearance of a lag phase in the reaction of the protein with iodoacetate after an incubation at alkaline pH. Such a lag phase does not appear when this incubation is carried out at neutral pH. Since this lag phase is similar to that which shows up in the activity during the isomerization of the enzyme from its alkaline to its neutral state, the present data are interpreted as implying a concerted movement of isoleucine‐16 and methionine‐192 during this isomerization process. They also indicate that in the alkaline form of the enzyme methionine‐192 has moved back into the interior of the protein. Since the spectroscopic properties of the zymogen and of the high‐pH form of the enzyme are similar they suggest that methionine‐192 occupies in the alkaline conformation of the enzyme a similar position as it does in the zymogen.