Zymogen Activation Confers Thermodynamic Stability on a Key Peptide Bond and Protects Human Cationic Trypsin from Degradation

András Szabó,Evette S Radisky,Miklós Sahin-Tóth

doi:10.1074/jbc.m113.538884

András Szabó, Evette S Radisky + Show 1 more

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https://doi.org/10.1074/jbc.m113.538884

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Abstract

Human cationic trypsinogen, precursor of the digestive enzyme trypsin, can be rapidly degraded to protect the pancreas when pathological conditions threaten, while trypsin itself is impressively resistant to degradation. For either form, degradation is controlled by two necessary initial proteolytic events: cleavage of the Leu81-Glu82 peptide bond by chymotrypsin C (CTRC) and cleavage of the Arg122-Val123 peptide bond by trypsin. Here we demonstrate that the Leu81-Glu82 peptide bond of human cationic trypsin, but not trypsinogen, is thermodynamically stable, such that cleavage by CTRC leads to an equilibrium mixture containing 10% cleaved and 90% uncleaved trypsin. When cleaved trypsin was incubated with CTRC, the Leu81-Glu82 peptide bond was re-synthesized to establish the same equilibrium. The thermodynamic stability of the scissile peptide bond was not dependent on CTRC or Leu-81, as re-synthesis was also accomplished by other proteases acting on mutated cationic trypsin. The Leu81-Glu82 peptide bond is located within a calcium binding loop, and thermodynamic stability of the bond was strictly dependent on calcium and on the calcium-coordinated residue Glu-85. Trypsinolytic cleavage of the Arg122-Val123 site was also delayed in trypsin relative to trypsinogen in a calcium-dependent manner, but for this bond cleavage was modulated by kinetic rather than thermodynamic control. Our results reveal that the trypsinogen to trypsin conformational switch modulates cleavage susceptibility of nick sites by altering both the thermodynamics and kinetics of cleavage to protect human cationic trypsin from premature degradation.

Highlights

Initial cleavage by chymotrypsin C regulates degradation of human cationic trypsin
Cleavage and Re-synthesis of the Leu81–Glu82 Peptide Bond in Human Cationic Trypsin by Chymotrypsin C—Previously we discovered that degradation of human cationic trypsin is regulated by chymotrypsin C (CTRC) cleavage at the Leu81–Glu82 bond (Fig. 1A), which is necessary for subsequent autolytic breakdown [6]
Incubation of cationic trypsin with a catalytic amount of CTRC in 1 mM calcium initially resulted in the appearance of products formed by cleavage at the Leu81–Glu82 peptide bond, but surprisingly the reaction did not proceed to completion; minimal (Ͻ10%) cleavage occurred within 10 min but no further progress was observed over the course of one hour (Fig. 1, B and D, left panel)

Summary

Introduction

Initial cleavage by chymotrypsin C regulates degradation of human cationic trypsin. For either form, degradation is controlled by two necessary initial proteolytic events: cleavage of the Leu81–Glu peptide bond by chymotrypsin C (CTRC) and cleavage of the Arg122–Val123 peptide bond by trypsin. We demonstrate that the Leu81–Glu peptide bond of human cationic trypsin, but not trypsinogen, is thermodynamically stable, such that cleavage by CTRC leads to an equilibrium mixture containing 10% cleaved and 90% uncleaved trypsin. Trypsinolytic cleavage of the Arg122–Val123 site was delayed in trypsin relative to trypsinogen in a calcium-dependent manner, but for this bond cleavage was modulated by kinetic rather than thermodynamic control. Our results reveal that the trypsinogen to trypsin conformational switch modulates cleavage susceptibility of nick sites by altering both the thermodynamics and kinetics of cleavage to protect human cationic trypsin from premature degradation

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