Effects of serpin binding on the target proteinase: Global stabilization, localized increased structural flexibility, and conserved hydrogen bonding at the active site

Gyula Kaslik, J. Kardos, Erika Szabó, László Szilágyi, P. Závodszky, William M. Westler, John L. Markley, L. Gráf

Research output: Contribution to journalArticle

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Abstract

The binding of human α1-proteinase inhibitor to rat trypsin was shown by NMR spectroscopy to raise the pK(a)' of His57 in the active site but not to disrupt the hydrogen bond between His57 and Asp102. Similar NMR results were observed for the Asp189 to serine mutant of rat trypsin, which is much more stable than wild-type trypsin against autoproteolysis as the result of mutation of the residue at the base of the specificity pocket. This mutant was used in further studies aimed at determining the extent of the conformational transition in trypsin that accompanies serpin binding and leads to disruption of the catalytic activity of the proteinase such that the inhibitor complex is trapped at the acyl enzyme intermediate stage. The stability of rat trypsin toward thermal denaturation was found to be lower in the free enzyme than in the complex with α1-proteinase inhibitor. This suggests that the complex contains extensive protein-protein interactions that stabilize overall folding. On the other hand, previous investigations have shown that the proteinase in serpin-proteinase complexes becomes more susceptible to limited proteolysis, suggesting that the conformational change that accompanies binding leads to the exposure of susceptible loops in the enzyme. The existence of this type of conformational change upon complex formation has been confirmed here by investigation of the rate of cleavage of disulfide linkages by added dithiothreitol. This study revealed that, despite the increased stability of trypsin in the complex, one or more of its disulfide bridges becomes much more easily reduced. We suggest that the process of complex formation with α1-proteinase inhibitor converts trypsin D189S into an inactive, loose structure, which serves as a 'conformational trap' of the enzyme that prevents catalytic deacylation. It is also proposed that plastic region(s) of the activation domain of trypsin may play a crucial role in this inhibitor-induced structural rearrangement.

Original languageEnglish
Pages (from-to)5455-5464
Number of pages10
JournalBiochemistry
Volume36
Issue number18
DOIs
Publication statusPublished - May 6 1997

Fingerprint

Serpins
Hydrogen Bonding
Trypsin
Catalytic Domain
Hydrogen bonds
Peptide Hydrolases
Stabilization
Rats
Enzymes
Disulfides
Proteolysis
Denaturation
Trypsin Inhibitors
Dithiothreitol
Serine
Nuclear magnetic resonance spectroscopy
Plastics
Hydrogen
Catalyst activity
Proteins

ASJC Scopus subject areas

  • Biochemistry

Cite this

Effects of serpin binding on the target proteinase : Global stabilization, localized increased structural flexibility, and conserved hydrogen bonding at the active site. / Kaslik, Gyula; Kardos, J.; Szabó, Erika; Szilágyi, László; Závodszky, P.; Westler, William M.; Markley, John L.; Gráf, L.

In: Biochemistry, Vol. 36, No. 18, 06.05.1997, p. 5455-5464.

Research output: Contribution to journalArticle

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abstract = "The binding of human α1-proteinase inhibitor to rat trypsin was shown by NMR spectroscopy to raise the pK(a)' of His57 in the active site but not to disrupt the hydrogen bond between His57 and Asp102. Similar NMR results were observed for the Asp189 to serine mutant of rat trypsin, which is much more stable than wild-type trypsin against autoproteolysis as the result of mutation of the residue at the base of the specificity pocket. This mutant was used in further studies aimed at determining the extent of the conformational transition in trypsin that accompanies serpin binding and leads to disruption of the catalytic activity of the proteinase such that the inhibitor complex is trapped at the acyl enzyme intermediate stage. The stability of rat trypsin toward thermal denaturation was found to be lower in the free enzyme than in the complex with α1-proteinase inhibitor. This suggests that the complex contains extensive protein-protein interactions that stabilize overall folding. On the other hand, previous investigations have shown that the proteinase in serpin-proteinase complexes becomes more susceptible to limited proteolysis, suggesting that the conformational change that accompanies binding leads to the exposure of susceptible loops in the enzyme. The existence of this type of conformational change upon complex formation has been confirmed here by investigation of the rate of cleavage of disulfide linkages by added dithiothreitol. This study revealed that, despite the increased stability of trypsin in the complex, one or more of its disulfide bridges becomes much more easily reduced. We suggest that the process of complex formation with α1-proteinase inhibitor converts trypsin D189S into an inactive, loose structure, which serves as a 'conformational trap' of the enzyme that prevents catalytic deacylation. It is also proposed that plastic region(s) of the activation domain of trypsin may play a crucial role in this inhibitor-induced structural rearrangement.",
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AU - Szilágyi, László

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N2 - The binding of human α1-proteinase inhibitor to rat trypsin was shown by NMR spectroscopy to raise the pK(a)' of His57 in the active site but not to disrupt the hydrogen bond between His57 and Asp102. Similar NMR results were observed for the Asp189 to serine mutant of rat trypsin, which is much more stable than wild-type trypsin against autoproteolysis as the result of mutation of the residue at the base of the specificity pocket. This mutant was used in further studies aimed at determining the extent of the conformational transition in trypsin that accompanies serpin binding and leads to disruption of the catalytic activity of the proteinase such that the inhibitor complex is trapped at the acyl enzyme intermediate stage. The stability of rat trypsin toward thermal denaturation was found to be lower in the free enzyme than in the complex with α1-proteinase inhibitor. This suggests that the complex contains extensive protein-protein interactions that stabilize overall folding. On the other hand, previous investigations have shown that the proteinase in serpin-proteinase complexes becomes more susceptible to limited proteolysis, suggesting that the conformational change that accompanies binding leads to the exposure of susceptible loops in the enzyme. The existence of this type of conformational change upon complex formation has been confirmed here by investigation of the rate of cleavage of disulfide linkages by added dithiothreitol. This study revealed that, despite the increased stability of trypsin in the complex, one or more of its disulfide bridges becomes much more easily reduced. We suggest that the process of complex formation with α1-proteinase inhibitor converts trypsin D189S into an inactive, loose structure, which serves as a 'conformational trap' of the enzyme that prevents catalytic deacylation. It is also proposed that plastic region(s) of the activation domain of trypsin may play a crucial role in this inhibitor-induced structural rearrangement.

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