Structural Basis for Accelerated Cleavage of Bovine Pancreatic Trypsin Inhibitor (BPTI) by Human Mesotrypsin

Moh'D A Salameh,Alexei S Soares,Alexandra Hockla,Evette S Radisky

doi:10.1074/jbc.m708268200

Moh'D A Salameh, Alexei S Soares + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m708268200

Copy DOI

Abstract

Human mesotrypsin is an isoform of trypsin that displays unusual resistance to polypeptide trypsin inhibitors and has been observed to cleave several such inhibitors as substrates. Whereas substitution of arginine for the highly conserved glycine 193 in the trypsin active site has been implicated as a critical factor in the inhibitor resistance of mesotrypsin, how this substitution leads to accelerated inhibitor cleavage is not clear. Bovine pancreatic trypsin inhibitor (BPTI) forms an extremely stable and cleavage-resistant complex with trypsin, and thus provides a rigorous challenge of mesotrypsin catalytic activity toward polypeptide inhibitors. Here, we report kinetic constants for mesotrypsin and the highly homologous (but inhibitor sensitive) human cationic trypsin, describing inhibition by, and cleavage of BPTI, as well as crystal structures of the mesotrypsin-BPTI and human cationic trypsin-BPTI complexes. We find that mesotrypsin cleaves BPTI with a rate constant accelerated 350-fold over that of human cationic trypsin and 150,000-fold over that of bovine trypsin. From the crystal structures, we see that small conformational adjustments limited to several side chains enable mesotrypsin-BPTI complex formation, surmounting the predicted steric clash introduced by Arg-193. Our results show that the mesotrypsin-BPTI interface favors catalysis through (a) electrostatic repulsion between the closely spaced mesotrypsin Arg-193 and BPTI Arg-17, and (b) elimination of two hydrogen bonds between the enzyme and the amine leaving group portion of BPTI. Our model predicts that these deleterious interactions accelerate leaving group dissociation and deacylation.

Highlights

There are three human trypsins encoded by different genes; cationic trypsinogen (PRSS1) and anionic trypsinogen (PRSS2) are located at proximal loci on chromosome 7q35, while mesotrypsinogen (PRSS3) is found on chromosome 9p13 [1]
We describe kinetic studies measuring the ability of mesotrypsin and human cationic trypsin to cleave Bovine pancreatic trypsin inhibitor (BPTI), as well as crystal structures of the mesotrypsin-BPTI and cationic trypsin-BPTI complexes
A 16-h time course shows the attainment of binding equilibrium by a series of parallel reactions with varying [BPTI] as indicated on the figure

Summary

EXPERIMENTAL PROCEDURES

Expression, Purification, and Activation of Recombinant Human Trypsins—Expression plasmids derived from pTrapT7, harboring genes for human cationic trypsinogen (PRSS1) [21] and human mesotrypsinogen (PRSS3) [14], were generous gifts from Dr Miklos Sahin-Toth (Boston University). Data were analyzed as described previously for a similar case of slow, tight-binding inhibition [28] For such a case, when inhibitor is in large excess over enzyme (as here), the relationship described by equation 2 is valid [28], where vi and v0 are the steady-state rates in the presence and absence of inhibitor, Km is the Michaelis constant for trypsin cleavage of Z-GPR-pNA, and [S0] and [I0] are the initial concentrations of substrate and. Search models were derived from previous structures of human cationic trypsin (PDB ID 1TRN, chain A) [36] and BPTI (PDB ID 2PTC, chain I) [37], and the successful solution contained 2 copies of each protein in the asymmetric unit, forming 2 canonical trypsin-BPTI complexes. Superpositions and Figures—All superpositions and structure figures were created using the graphics software Pymol [43]

RESULTS

Human mesotrypsin

DISCUSSION