Abstract
Acetylation of Pronase Guanidine Stable Chymoelastase in 50% glycerol results in modification of both the (alpha)-amino group of isoleucine 1 and the (epsilon)-amino group of the single lysine residue with retention of activity. The pH-rate profile of inhibition with chloromethylketones is also unchanged.The group in chymotrypsin that governs the activity with specific substrates at alkaline pH is believed to be the amino group of the amino-terminal isoleucine. In order to re-examine the role of the amino group in chymotrypsin activity we first attempted chemical modification with acetic anhydride, a technique that has been used extensively in elucidating the role of various groups in proteins, including chymotrypsin. We have found that glycerol prevents the loss of activity upon acetylation by protecting the alpha amino group. Modification of the amino group with methylacetimidate has been shown to abolish the activity of delta chymotrypsin. Guanidination of lysine amino groups prior to amidination protects against this loss.The role of glycerol or guanidination in stabilizing chymotrypsin has been shown in studies with proflavin to be exerted through a shift in the equilibrium between the active and inactive forms of the enzyme so that at any pH the fraction of enzyme in the active conformation is increased.The pKa of the alpha amino group depends on the extent to which it is buried in a salt bridge, thus glycerol or guanidination provide a way of "modifying" the alpha amino group. Their effect on the titration behavior of this group has been studied. These techniques can be used to shift the apparent pKa of the alpha amino group, allowing one to study the importance of this group in the activity of chymotrypsin at alkaline pH. Our data indicate that the alpha amino group may not be completely responsible for the loss of activity of chymotrypsin at alkaline pH.