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W2019936286 abstract "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 β-trypsin in the acidic pH range yielded substantial differences in the stability parameters. Compared to β-trypsin, trypsinogen exhibits lower enthalpy of denaturation ΔHden, higher denaturational heat capacity change ΔCp,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 ΔGden = 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 ΔGden = 3.2 kcal/mole, but increases by ΔΔGden = 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 HiBovine 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 β-trypsin in the acidic pH range yielded substantial differences in the stability parameters. Compared to β-trypsin, trypsinogen exhibits lower enthalpy of denaturation ΔHden, higher denaturational heat capacity change ΔCp,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 ΔGden = 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 ΔGden = 3.2 kcal/mole, but increases by ΔΔGden = 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 HiBovine 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 β-trypsin in the acidic pH range yielded substantial differences in the stability parameters. Compared to β-trypsin, trypsinogen exhibits lower enthalpy of denaturation ΔHden, higher denaturational heat capacity change ΔCp,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 ΔGden = 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 ΔGden = 3.2 kcal/mole, but increases by ΔΔGden = 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 HiBovine 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 β-trypsin in the acidic pH range yielded substantial differences in the stability parameters. Compared to β-trypsin, trypsinogen exhibits lower enthalpy of denaturation ΔHden, higher denaturational heat capacity change ΔCp,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 ΔGden = 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 ΔGden = 3.2 kcal/mole, but increases by ΔΔGden = 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 β-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.0-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 β-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 HiBovine 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 β-trypsin in the acidic pH range yielded substantial differences in the stability parameters. Compared to β-trypsin, trypsinogen exhibits lower enthalpy of denaturation ΔHden, higher denaturational heat capacity change ΔCp,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 ΔGden = 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 ΔGden = 3.2 kcal/mole, but increases by ΔΔGden = 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 β-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.0 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.0-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 β-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 HiBovine 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 β-trypsin in the acidic pH range yielded substantial differences in the stability parameters. Compared to β-trypsin, trypsinogen exhibits lower enthalpy of denaturation ΔHden, higher denaturational heat capacity change ΔCp,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 ΔGden = 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 ΔGden = 3.2 kcal/mole, but increases by ΔΔGden = 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 HiBovine 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 β-trypsin in the acidic pH range yielded substantial differences in the stability parameters. Compared to β-trypsin, trypsinogen exhibits lower enthalpy of denaturation ΔHden, higher denaturational heat capacity change ΔCp,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 ΔGden = 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 ΔGden = 3.2 kcal/mole, but increases by ΔΔGden = 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 β-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.0-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 β-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 HiBovine 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 β-trypsin in the acidic pH range yielded substantial differences in the stability parameters. Compared to β-trypsin, trypsinogen exhibits lower enthalpy of denaturation ΔHden, higher denaturational heat capacity change ΔCp,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 ΔGden = 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 ΔGden = 3.2 kcal/mole, but increases by ΔΔGden = 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 β-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.0 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.0 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.0-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 β-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 HiBovine 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 β-trypsin in the acidic pH range yielded substantial differences in the stability parameters. Compared to β-trypsin, trypsinogen exhibits lower enthalpy of denaturation ΔHden, higher denaturational heat capacity change ΔCp,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 ΔGden = 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 ΔGden = 3.2 kcal/mole, but increases by ΔΔGden = 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 HiBovine 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 β-trypsin in the acidic pH range yielded substantial differences in the stability parameters. Compared to β-trypsin, trypsinogen exhibits lower enthalpy of denaturation ΔHden, higher denaturational heat capacity change ΔCp,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 ΔGden = 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 ΔGden = 3.2 kcal/mole, but increases by ΔΔGden = 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 HiBovine 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 β-trypsin in the acidic pH range yielded substantial differences in the stability parameters. Compared to β-trypsin, trypsinogen exhibits lower enthalpy of denaturation ΔHden, higher denaturational heat capacity change ΔCp,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 ΔGden = 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 ΔGden = 3.2 kcal/mole, but increases by ΔΔGden = 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 β-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.0-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 β-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 HiBovine 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 β-trypsin in the acidic pH range yielded substantial differences in the stability parameters. Compared to β-trypsin, trypsinogen exhibits lower enthalpy of denaturation ΔHden, higher denaturational heat capacity change ΔCp,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 ΔGden = 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 ΔGden = 3.2 kcal/mole, but increases by ΔΔGden = 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 β-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.0 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.0-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 β-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 HiBovine 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 β-trypsin in the acidic pH range yielded substantial differences in the stability parameters. Compared to β-trypsin, trypsinogen exhibits lower enthalpy of denaturation ΔHden, higher denaturational heat capacity change ΔCp,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 ΔGden = 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 ΔGden = 3.2 kcal/mole, but increases by ΔΔGden = 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 HiBovine 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 β-trypsin in the acidic pH range yielded substantial differences in the stability parameters. Compared to β-trypsin, trypsinogen exhibits lower enthalpy of denaturation ΔHden, higher denaturational heat capacity change ΔCp,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 ΔGden = 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 ΔGden = 3.2 kcal/mole, but increases by ΔΔGden = 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 β-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.0-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 β-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 HiBovine 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 β-trypsin in the acidic pH range yielded substantial differences in the stability parameters. Compared to β-trypsin, trypsinogen exhibits lower enthalpy of denaturation ΔHden, higher denaturational heat capacity change ΔCp,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 ΔGden = 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 ΔGden = 3.2 kcal/mole, but increases by ΔΔGden = 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 β-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.0 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.0 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.0 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." @default.
W2019936286 created "2016-06-24" @default.
W2019936286 creator A5065096705 @default.
W2019936286 creator A5079071978 @default.
W2019936286 date "1995-04-01" @default.
W2019936286 modified "2023-10-03" @default.
W2019936286 title "Ligand-induced changes in the conformational stability of bovine trypsinogen and their implications for the protein function" @default.
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W2019936286 doi "https://doi.org/10.1016/s0022-2836(05)80149-4" @default.
W2019936286 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/7723025" @default.
W2019936286 hasPublicationYear "1995" @default.
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