Human cationic trypsinogen. Arg(117) is the reactive site of an inhibitory surface loop that controls spontaneous zymogen activation.
Mutation of Arg(117), 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 Arg(117) was investigated in wild-type cationic Tg and in the activation-resistant Lys(15) --> Gln mutant (K15Q-Tg), in which Tg-specific properties of Arg(117) can be examined selectively. We found that trypsinolytic cleavage of the Arg(117)-Val(118) 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 Ca(2+), at pH 8.0, the hydrolysis equilibrium (K(hyd) = [cleaved Tg]/[intact Tg]) was 5.4, whereas 5 mm Ca(2+) reduced the rate of cleavage at Arg(117) at least 20-fold, and shifted K(hyd) to 0.7. These observations indicate that the Arg(117)-Val(118) 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 K(i) of 80 microm, while no inhibition was observed with K15Q-Tg carrying the Arg(117) --> His mutation. Finally, zymogen breakdown due to other trypsinolytic pathways was shown to proceed almost 2000-fold slower than cleavage at Arg(117). 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 Arg(117) results in inhibition of trypsin, whereas high trypsin concentrations degrade Tg, thus limiting further zymogen activation. Loss of Arg(117)-dependent trypsin inhibition can contribute to the development of hereditary pancreatitis associated with the Arg(117) --> His mutation.
Kukor
Zoltán
Z
Department of Physiology, University of California Los Angeles, Los Angeles, California 90095-1662, USA.
Tóth
Miklós
M
Pál
Gábor
G
Sahin-Tóth
Miklós
M
D019943:Amino Acid Substitution; D001120:Arginine; D001665:Binding Sites; D019281:Dimerization; D004792:Enzyme Precursors / Q000737:chemistry*; D006801:Humans; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D010195:Pancreatitis / Q000201:enzymology / Q000235:genetics; D014362:Trypsinogen / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics
D004792:Enzyme Precursors; D014362:Trypsinogen; D001120:Arginine