Properties of the His57-Asp102 dyad of rat trypsin D189s in the zymogen, activated enzyme, and α1-proteinase inhibitor complexed forms

Gyula Kaslik, William M. Westler, L. Gráf, John L. Markley

Research output: Contribution to journalArticle

16 Citations (Scopus)

Abstract

Structural and biochemical studies suggest that serpins induce structural rearrangements in their target serine-proteinases. Previous NMR studies of the complex between a serpin, α1-proteinase inhibitor, and a mutant of recombinant rat trypsin (the Asp189 to Ser mutant, D189S, which is much more stable than wild-type rat trypsin against autoproteolysis) provided information about the state of catalytic residues in this complex: the hydrogen bond between Asp120 and His57 remains intact in the complex, and spectral properties of His57 are more like those of the zymogen than of the activated enzyme (G. Kaslik, et al., 1997, Biochemistry 36, 5455-5464). Here we report the protonation and exchange behavior of His57 of recombinant rat trypsin D189S in three states: the zymogen, the active enzyme, and the complex with human α1-proteinase inhibitor and compare these with analogous behavior of His57 of bovine chymotrypsinogen and α-chymotrypsin. In these studies the pK(a) of His57 has been determined from the pH dependence of the 1H NMR signal from the H(δ1) proton of histidine in the Asp102-His57 dyad, and a measure of the accessibility of this part of the active site has been obtained from the rate of appearance of this signal following its selective saturation. The activation of rat trypsinogen D189S (zymogen, pK(a) = 7.8 ± 0.1; Hill coefficient = 0.86 ± 0.05) decreased the pK(a) of His57 by 1.1 unit and made the protonation process cooperative (active enzyme, pK(a) = 6.7 ± 0.1; Hill coefficient = 1.37 ± 0.08). The binding of α1-proteinase inhibitor to trypsin D189S led to an increase in the pK(a) value of His57 to a value higher than that of the zymogen and led to negative cooperativity in the protonation process (complex, pK(a) = 8.1 ± 0.1; Hill coefficient = 0.70 ± 0.08), as was observed for the zymogen. In spite of these differences in the pK(a) of His57 in the zymogen, active enzyme, and α1-proteinase inhibitor complex, the solvent exchange lifetime of the His57 H(δ1) proton was the same, within experimental error, in all three states (lifetime = 2 to 12.5 ms). The linewidth of the 1H NMR signal from the H(δ1) proton of His57 was relatively sharp, at temperatures between 5 and 20°C at both low pH (5.2) and high pH (10.0), in spectra of bovine α-chymotrypsin, recombinant rat trypsin D189S, and the complex between rat trypsin D189S and human α1- proteinase inhibitor; however, in spectra of the complex between α- chymotrypsin and human α1-proteinase inhibitor, the peak was broader and could be well-resolved only at the lower temperature (5°C).

Original languageEnglish
Pages (from-to)254-264
Number of pages11
JournalArchives of Biochemistry and Biophysics
Volume362
Issue number2
DOIs
Publication statusPublished - Feb 15 1999

Fingerprint

Enzyme Precursors
Trypsin
Rats
Peptide Hydrolases
Protonation
Chymotrypsin
Enzymes
Serpins
Protons
Nuclear magnetic resonance
Chymotrypsinogen
Trypsinogen
Biochemistry
Temperature
Trypsin Inhibitors
Serine Proteases
Histidine
Linewidth
Hydrogen
Catalytic Domain

Keywords

  • Active site
  • NMR
  • Serine-proteases
  • Serpin

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

Cite this

Properties of the His57-Asp102 dyad of rat trypsin D189s in the zymogen, activated enzyme, and α1-proteinase inhibitor complexed forms. / Kaslik, Gyula; Westler, William M.; Gráf, L.; Markley, John L.

In: Archives of Biochemistry and Biophysics, Vol. 362, No. 2, 15.02.1999, p. 254-264.

Research output: Contribution to journalArticle

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abstract = "Structural and biochemical studies suggest that serpins induce structural rearrangements in their target serine-proteinases. Previous NMR studies of the complex between a serpin, α1-proteinase inhibitor, and a mutant of recombinant rat trypsin (the Asp189 to Ser mutant, D189S, which is much more stable than wild-type rat trypsin against autoproteolysis) provided information about the state of catalytic residues in this complex: the hydrogen bond between Asp120 and His57 remains intact in the complex, and spectral properties of His57 are more like those of the zymogen than of the activated enzyme (G. Kaslik, et al., 1997, Biochemistry 36, 5455-5464). Here we report the protonation and exchange behavior of His57 of recombinant rat trypsin D189S in three states: the zymogen, the active enzyme, and the complex with human α1-proteinase inhibitor and compare these with analogous behavior of His57 of bovine chymotrypsinogen and α-chymotrypsin. In these studies the pK(a) of His57 has been determined from the pH dependence of the 1H NMR signal from the H(δ1) proton of histidine in the Asp102-His57 dyad, and a measure of the accessibility of this part of the active site has been obtained from the rate of appearance of this signal following its selective saturation. The activation of rat trypsinogen D189S (zymogen, pK(a) = 7.8 ± 0.1; Hill coefficient = 0.86 ± 0.05) decreased the pK(a) of His57 by 1.1 unit and made the protonation process cooperative (active enzyme, pK(a) = 6.7 ± 0.1; Hill coefficient = 1.37 ± 0.08). The binding of α1-proteinase inhibitor to trypsin D189S led to an increase in the pK(a) value of His57 to a value higher than that of the zymogen and led to negative cooperativity in the protonation process (complex, pK(a) = 8.1 ± 0.1; Hill coefficient = 0.70 ± 0.08), as was observed for the zymogen. In spite of these differences in the pK(a) of His57 in the zymogen, active enzyme, and α1-proteinase inhibitor complex, the solvent exchange lifetime of the His57 H(δ1) proton was the same, within experimental error, in all three states (lifetime = 2 to 12.5 ms). The linewidth of the 1H NMR signal from the H(δ1) proton of His57 was relatively sharp, at temperatures between 5 and 20°C at both low pH (5.2) and high pH (10.0), in spectra of bovine α-chymotrypsin, recombinant rat trypsin D189S, and the complex between rat trypsin D189S and human α1- proteinase inhibitor; however, in spectra of the complex between α- chymotrypsin and human α1-proteinase inhibitor, the peak was broader and could be well-resolved only at the lower temperature (5°C).",
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T1 - Properties of the His57-Asp102 dyad of rat trypsin D189s in the zymogen, activated enzyme, and α1-proteinase inhibitor complexed forms

AU - Kaslik, Gyula

AU - Westler, William M.

AU - Gráf, L.

AU - Markley, John L.

PY - 1999/2/15

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N2 - Structural and biochemical studies suggest that serpins induce structural rearrangements in their target serine-proteinases. Previous NMR studies of the complex between a serpin, α1-proteinase inhibitor, and a mutant of recombinant rat trypsin (the Asp189 to Ser mutant, D189S, which is much more stable than wild-type rat trypsin against autoproteolysis) provided information about the state of catalytic residues in this complex: the hydrogen bond between Asp120 and His57 remains intact in the complex, and spectral properties of His57 are more like those of the zymogen than of the activated enzyme (G. Kaslik, et al., 1997, Biochemistry 36, 5455-5464). Here we report the protonation and exchange behavior of His57 of recombinant rat trypsin D189S in three states: the zymogen, the active enzyme, and the complex with human α1-proteinase inhibitor and compare these with analogous behavior of His57 of bovine chymotrypsinogen and α-chymotrypsin. In these studies the pK(a) of His57 has been determined from the pH dependence of the 1H NMR signal from the H(δ1) proton of histidine in the Asp102-His57 dyad, and a measure of the accessibility of this part of the active site has been obtained from the rate of appearance of this signal following its selective saturation. The activation of rat trypsinogen D189S (zymogen, pK(a) = 7.8 ± 0.1; Hill coefficient = 0.86 ± 0.05) decreased the pK(a) of His57 by 1.1 unit and made the protonation process cooperative (active enzyme, pK(a) = 6.7 ± 0.1; Hill coefficient = 1.37 ± 0.08). The binding of α1-proteinase inhibitor to trypsin D189S led to an increase in the pK(a) value of His57 to a value higher than that of the zymogen and led to negative cooperativity in the protonation process (complex, pK(a) = 8.1 ± 0.1; Hill coefficient = 0.70 ± 0.08), as was observed for the zymogen. In spite of these differences in the pK(a) of His57 in the zymogen, active enzyme, and α1-proteinase inhibitor complex, the solvent exchange lifetime of the His57 H(δ1) proton was the same, within experimental error, in all three states (lifetime = 2 to 12.5 ms). The linewidth of the 1H NMR signal from the H(δ1) proton of His57 was relatively sharp, at temperatures between 5 and 20°C at both low pH (5.2) and high pH (10.0), in spectra of bovine α-chymotrypsin, recombinant rat trypsin D189S, and the complex between rat trypsin D189S and human α1- proteinase inhibitor; however, in spectra of the complex between α- chymotrypsin and human α1-proteinase inhibitor, the peak was broader and could be well-resolved only at the lower temperature (5°C).

AB - Structural and biochemical studies suggest that serpins induce structural rearrangements in their target serine-proteinases. Previous NMR studies of the complex between a serpin, α1-proteinase inhibitor, and a mutant of recombinant rat trypsin (the Asp189 to Ser mutant, D189S, which is much more stable than wild-type rat trypsin against autoproteolysis) provided information about the state of catalytic residues in this complex: the hydrogen bond between Asp120 and His57 remains intact in the complex, and spectral properties of His57 are more like those of the zymogen than of the activated enzyme (G. Kaslik, et al., 1997, Biochemistry 36, 5455-5464). Here we report the protonation and exchange behavior of His57 of recombinant rat trypsin D189S in three states: the zymogen, the active enzyme, and the complex with human α1-proteinase inhibitor and compare these with analogous behavior of His57 of bovine chymotrypsinogen and α-chymotrypsin. In these studies the pK(a) of His57 has been determined from the pH dependence of the 1H NMR signal from the H(δ1) proton of histidine in the Asp102-His57 dyad, and a measure of the accessibility of this part of the active site has been obtained from the rate of appearance of this signal following its selective saturation. The activation of rat trypsinogen D189S (zymogen, pK(a) = 7.8 ± 0.1; Hill coefficient = 0.86 ± 0.05) decreased the pK(a) of His57 by 1.1 unit and made the protonation process cooperative (active enzyme, pK(a) = 6.7 ± 0.1; Hill coefficient = 1.37 ± 0.08). The binding of α1-proteinase inhibitor to trypsin D189S led to an increase in the pK(a) value of His57 to a value higher than that of the zymogen and led to negative cooperativity in the protonation process (complex, pK(a) = 8.1 ± 0.1; Hill coefficient = 0.70 ± 0.08), as was observed for the zymogen. In spite of these differences in the pK(a) of His57 in the zymogen, active enzyme, and α1-proteinase inhibitor complex, the solvent exchange lifetime of the His57 H(δ1) proton was the same, within experimental error, in all three states (lifetime = 2 to 12.5 ms). The linewidth of the 1H NMR signal from the H(δ1) proton of His57 was relatively sharp, at temperatures between 5 and 20°C at both low pH (5.2) and high pH (10.0), in spectra of bovine α-chymotrypsin, recombinant rat trypsin D189S, and the complex between rat trypsin D189S and human α1- proteinase inhibitor; however, in spectra of the complex between α- chymotrypsin and human α1-proteinase inhibitor, the peak was broader and could be well-resolved only at the lower temperature (5°C).

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