Reaction of acetic and maleic anhydrides with elastase. Evidence for a role of the amino-terminal valine

Porcine pancreatic elastase treated with acetic anhydride in the neutral pH range was reversibly inactivated. At room temperature and pH 7.6 the enzyme was reactivated over a period of about 1 hr. At pH 9.5 reactivation was complete in less than 1 min. All three lysine residues and 6.03 ± 0.5 tyrosine residues were acetylated while the NH₂-terminal valine remained unsubstituted. The group responsible for inactivation has not been identified, but lysine and tyrosine have been ruled out. Hypothetically it is suggested that acetylation of histidine- 57 is responsible for the observed effects. At pH 10.5, or at pH 9-9.5 in 2 and 4 M urea, acetic anhydride irreversibly inactivated elastase. There was a direct correlation between the residual activity of partially inactivated elastase and the residual unblocked NH₂-terminal group. Unsubstituted valine-16 was found entirely in the active part of partially inactivated enzyme. Inactive elastase was devoid of a free NH₂-terminal group. Treatment of elastase with maleic anhydride at pH 11.5 caused loss of esterase activity and corresponding loss of free NH₂-terminal valine. Differences in the circular dichroism spectrum between pH 8.5 and 10.6 were compared with differences between pH 4.5 and 2.9 where a conformational change involving the NH₂-terminal valine has previously been demonstrated. The magnitude of the changes and the wavelengths where they occurred were similar in the high and the low pH ranges. Therefore the CD changes occurring between pH 8.5 and 10.6 are indicative of, or at least compatible with, a conformational change in the NH₂-terminal region. The results suggest that the free amino group of valine-16 is required for full enzymatic activity, and in analogy with other serine proteases probably maintains the active conformation by forming an ion pair with aspartic acid-194.