title: Human cationic trypsinogen. Role of Asn-21 in zymogen activation and implications in hereditary pancreatitis.
abstract: Mutation Asn-21 --> Ile in human cationic trypsinogen (Tg-1) has been associated with hereditary pancreatitis. Recent studies with rat anionic Tg (Tg-2) indicated that the analogous Thr-21 --> Ile mutation stabilizes the zymogen against autoactivation, whereas it has no effect on catalytic properties or autolytic stability of trypsin (Sahin-Tóth, M. (1999) J. Biol. Chem. 274, 29699-29704). In the present paper, human cationic Tg (Asn-21-Tg) and mutants Asn-21 --> Ile (Ile-21-Tg) and Asn-21 --> Thr (Thr-21-Tg) were expressed in Escherichia coli, and zymogen activation, zymogen degradation, and trypsin autolysis were studied. Enterokinase activated Asn-21-Tg approximately 2-fold better than Ile-21-Tg or Thr-21-Tg, and catalytic parameters of trypsins were comparable. At 37 degrees C, in 5 mm Ca(2+), all three trypsins were highly stable. In the absence of Ca(2+), Asn-21- and Ile-21-trypsins suffered autolysis in an indistinguishable manner, whereas Thr-21-trypsin exhibited significantly increased stability. In sharp contrast to observations with the rat proenzyme, at pH 8.0, 37 degrees C, autoactivation kinetics of Asn-21-Tg and Ile-21-Tg were identical; however, at pH 5. 0, Ile-21-Tg autoactivated at an enhanced rate relative to Asn-21-Tg. Remarkably, at both pH values, Thr-21-Tg showed markedly higher autoactivation rates than the two other zymogens. Finally, autocatalytic proteolysis of human zymogens was limited to cleavage at Arg-117, and no digestion at Lys-188 was detected. The observations indicate that zymogen stabilization by Ile-21 as observed in rat Tg-2 is not characteristic of human Tg-1. Instead, an increased propensity to autoactivation under acidic conditions might be relevant to the pathomechanism of the Asn-21 --> Ile mutation in hereditary pancreatitis. In the same context, faster autoactivation and increased trypsin stability caused by the Asn-21 --> Thr mutation in human Tg-1 might provide a rationale for the evolutionary divergence from Thr-21 found in other mammalian trypsinogens.
authors.affiliation: Department of Physiology, University of California, Los Angeles, California 90095-1662, USA. miklos@hhmi.ucla.edu
authors.forename: M
authors.initials: M
authors.lastname: Sahin-Tóth
mesh_terms: D000818:Animals; D001216:Asparagine / Q000378:metabolism*; D002384:Catalysis; D002412:Cations; D004765:Enteropeptidase / Q000378:metabolism; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000378:metabolism; D004795:Enzyme Stability; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D016297:Mutagenesis, Site-Directed; D010195:Pancreatitis / Q000201:enzymology* / Q000235:genetics; D051381:Rats; D014362:Trypsinogen / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D002412:Cations; D004792:Enzyme Precursors; D001216:Asparagine; D014362:Trypsinogen; D004765:Enteropeptidase