Human cationic trypsinogen. Arg117 is the reactive site of an inhibitory surface loop that controls spontaneous zymogen activation

Zoltán Kukor, Miklós Tóth, Gábor Pál, Miklós Sahin-Tóth

Research output: Contribution to journalArticle

54 Citations (Scopus)

Abstract

Mutation of Arg117, an autocatalytic cleavage site, is the most frequent amino acid change found in the cationic trypsinogen (Tg) of patients with hereditary pancreatitis. In the present study, the role of Arg117 was investigated in wild-type cationic Tg and in the activation-resistant Lys15 → Gln mutant (K15Q-Tg), in which Tg-specific properties of Arg117 can be examined selectively. We found that trypsinolytic cleavage of the Arg117-Val118 bond did not proceed to completion, but due to trypsin-catalyzed re-synthesis an equilibrium was established between intact Tg and its cleaved, two-chain form. In the absence of Ca2+, at pH 8.0, the hydrolysis equilibrium (Khyd = [cleaved Tg]/[intact Tg]) was 5.4, whereas 5 mM Ca2+ reduced the rate of cleavage at Arg117 at least 20-fold, and shifted Khyd to 0.7. These observations indicate that the Arg117-Val118 bond exhibits properties analogous to the reactive site bond of canonical trypsin inhibitors and suggest that this surface loop might serve as a low affinity inhibitor of zymogen activation. Consistent with this notion, autoactivation of cationic Tg was inhibited by the cleaved form of K15Q-Tg, with an estimated Ki of 80 μM, while no inhibition was observed with K15Q-Tg carrying the Arg117 → His mutation. Finally, zymogen breakdown due to other trypsinolytic pathways was shown to proceed almost 2000-fold slower than cleavage at Arg117. Taken together, the findings suggest two independent, successively functional trypsin-mediated mechanisms against pathological Tg activation in the pancreas. At low trypsin concentrations, cleavage at Arg117 results in inhibition of trypsin, whereas high trypsin concentrations degrade Tg, thus limiting further zymogen activation. Loss of Arg117-dependent trypsin inhibition can contribute to the development of hereditary pancreatitis associated with the Arg117 → His mutation.

Original languageEnglish
Pages (from-to)6111-6117
Number of pages7
JournalJournal of Biological Chemistry
Volume277
Issue number8
DOIs
Publication statusPublished - Feb 22 2002

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Fingerprint Dive into the research topics of 'Human cationic trypsinogen. Arg<sup>117</sup> is the reactive site of an inhibitory surface loop that controls spontaneous zymogen activation'. Together they form a unique fingerprint.

  • Cite this