Bovine trypsinogen was used as a model protein for studying changes in the conformational stability induced by pH or binding of the calcium ion. Spectrophotometrically monitored thermal unfolding of trypsinogen and beta-trypsin in the acidic pH range yielded substantial differences in the stability parameters. Compared to beta-trypsin, trypsinogen exhibits lower enthalpy of denaturation delta Hden, higher denaturational heat capacity change delta Cp,den, but very similar temperature of denaturation Tden. pH-dependence of the conformational stability of the ligand-free trypsinogen, measured also by GdnCl-induced unfolding, is bell shaped with the maximum free energy of unfolding delta Gden = 10.9 kcal/mole at pH 5.5 (4.5 pH units below its isoelectric point). At pH 8.3 the conformational stability of the zymogen drops to delta Gden = 3.2 kcal/mole, but increases by delta delta Gden = 6.1 kcal/mole in the presence of Ca2+. This significant stabilization of the zymogen by the calcium ion is also pH-dependent. To assess the effect of Ca2+ on the trypsinogen molecule, the spectrophotometric titrations and NOESY spectra were carried out. Based on the structural analysis, the long range effects between Ca2+-->Ile73-->Trp141 and the interdomain His40-Asp194 ion pair are proposed to be partially responsible for trypsinogen stabilization. Additionally, the steady-state parameters for hydrolysis of the oligopeptide amide substrate catalysed by free trypsinogen, its complexes with Ca2+ and the IleVal dipeptide and by beta-trypsin were measured. It appears that in the pH range 5.5 to 8.3 the stability and the catalytic activity/ligand binding properties are fully separated. Whereas the deprotonation of His57 accounts for the increase of kcat/km parameter, deprotonation of His40 is involved in the huge decrease of the conformational stability. Similarly, a large stabilization by the calcium ion is not accompanied by changes in enzymatic activity. Presented data are encouraging for an enzyme design directed toward improved stability. Study holds ProTherm entries: 7485, 7486, 7487, 7488, 7489 Extra Details: trypsinogen; stability change; calcium; enzyme catalysis;,ligand binding
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:34 p.m.
|Number of data points||15|
|Proteins||Cationic trypsin ; Anionic trypsin ; Cationic trypsin ; Cationic trypsin|
|Assays/Quantities/Protocols||Experimental Assay: Cm buffers:Tris-HCl: 0.1 M, pH:8.3 ; Experimental Assay: m buffers:Tris-HCl: 0.1 M, pH:8.3 ; Experimental Assay: dG_H2O buffers:Tris-HCl: 0.1 M, pH:8.3 ; Experimental Assay: Cm pH:7.5, buffers:Tris-HCl: 0.1 M ; Experimental Assay: m pH:7.5, buffers:Tris-HCl: 0.1 M ; Experimental Assay: dG_H2O pH:7.5, buffers:Tris-HCl: 0.1 M ; Experimental Assay: Cm pH:5.8, buffers:MES: 30 mM ; Experimental Assay: m pH:5.8, buffers:MES: 30 mM ; Experimental Assay: dG_H2O pH:5.8, buffers:MES: 30 mM ; Experimental Assay: dCp ; Experimental Assay: Tm ; Experimental Assay: dHvH|
|Libraries||Mutations for sequence IVGGYTCGANTVPYQVSLNSGYHFCGGSLINSQWVVSAAHCYKSGIQVRLGEDNINVVEGNEQFISASKSIVHPSYNSNTLNNDIMLIKLKSAASLNSRVASISLPTSCASAGTQCLISGWGNTKSSGTSYPDVLKCLKAPILSDSSCKSAYPGQITSNMFCAGYLEGGKDSCQGDSGGPVVCSGKLQGIVSWGSGCAQKNKPGVYTKVCNYVSWIKQTIASN ; Mutations for sequence VDDDDKIVGGYTCGANTVPYQVSLNSGYHFCGGSLINSQWVVSAAHCYKSGIQVRLGEDNINVVEGNEQFISASKSIVHPSYNSNTLNNDIMLIKLKSAASLNSRVASISLPTSCASAGTQCLISGWGNTKSSGTSYPDVLKCLKAPILSDSSCKSAYPGQITSNMFCAGYLEGGKDSCQGDSGGPVVCSGKLQGIVSWGSGCAQKNKPGVYTKVCNYVSWIKQTIASN|