Modification of a Single Disulfide Bond in Trypsinogen and the Activation of the Carboxymethyl Derivative

Light, Albert; Hardwick, Ben C.; Hatfield, Lon M.; Sondack, David L.

doi:10.1016/s0021-9258(18)63535-5

Cited by 29 publications

(5 citation statements)

References 23 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Two possible mechanisms were considered: (pathway a) a multiple interchange (solid arrows) involving Cysn-Cys38 (PTI) and both Cysm-Cysíos and Cys3i-Cys47 of trypsin; (pathway b) a simple interchange involving only CysH-Cys38 (PTI) and Cysi79-Cys203 (trypsin) (broken arrow). These 2 disulfide bridges have been found to be unusually reactive as compared to others (Kress and Laskowski, 1967;Light et al, 1969). The final mechanism will have to wait for further X-ray crystallography data with the trypsin-PTI complex.…”

Section: Discussionmentioning

confidence: 91%

“…The Cysi79-Cys2o3 bridge of trypsin can also be selectively reduced and alkylated (RCOM*trypsin) (Light et al, 1969;Hatfield et al, 1971) without destruction of the active site. Again stoichiometric association with PTI persists but the strength of the association decreases considerably.…”

Section: Discussionmentioning

confidence: 99%

“…The reduced derivative was then alkylated with iodoacetic acid and activated into RCOM*trypsin. All these operations were carried out by the techniques developed in Light's group (Light et al, 1969). Titrations of the active site were carried out with NPGB (Chase and Shaw, 1967).…”

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

Trypsin-pancreatic trypsin inhibitor association. Dynamics of the interaction and role of disulfide bridges

Vincent¹,

Lazdunski²

1972

Biochemistry

264

179

View full text Add to dashboard Cite

Section: Discussionmentioning

confidence: 91%

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Trypsin-pancreatic trypsin inhibitor association. Dynamics of the interaction and role of disulfide bridges

Vincent¹,

Lazdunski²

1972

Biochemistry

264

179

View full text Add to dashboard Cite

“…At 0°, pH 9.0, the Cys3o-Cys34 bridge in neurotoxin III is reduced by sodium borohydride (k = 1.7 X 10-2 M~l sec-1) 8-30 times faster than models such as oxytocin, cystine, or glutathione (k = 0.5-2 X 10-3 M~] sec-1 (Light et al, 1969)) and nearly as rapidly as the Cysi4-Cys3g bridge of the pancreatic trypsin inhibitor (k = 3.0 X 10-2 A/-1 sec-1 (unpublished result from this laboratory)) which is known from X-ray crystallographic data to be in a very exposed position at the protein inhibitor surface (Huber et al, 1971).…”

Section: Resultsmentioning

confidence: 99%

Structure-function relation in the binding of snake neurotoxins to the Torpedo membrane receptor

Chicheportiche¹,

Vincent²,

Kopeyan³

et al. 1975

Biochemistry

109

View full text Add to dashboard Cite

The Cys30-Cus34 bridge present in all long neutotoxins (71-74 amino acids, 5 disulfide bridges), but not in short toxins (60-63 amino acids, 4 disulfide bridges), is exposed at the surface since it can be reduced rapidly and selectively by sodium borohydride. Reduction and alkylation of the Cys30-Cys34 bridge of Naja haje neurotoxin III hardly alter the conformational properties of this model long toxin. Although alkylation by iodoacetic acid of th -SH groups liberated by reduction abolishes the toxicity, alkylation by iodoacetamide or ethylenimine does not affect the curarizing efficacy of the toxin. The Cys30-Cys34 bridge is not very important for the toxic activity of long neurotoxins. Reduction of the Cys30-Cys34 bridge followed by alkylation with radioactive iodoacetamide gave a labeled and active toxin which is a convenient derivative for binding experiments to the toxin receptor in membranes of the Torpedo electric organ. The binding capacity of these membrane is 1200 pmol of toxin/mg of membrane protein. The dissociation constant of the modified toxin-receptor complex at pH 7.4, 20 degrees is 10 minus 8m. Reduction with carbroxamidomethylation of the Cys30-Cys34 bridge decreases the affinity of the native Naja haje toxin only by a factor of 15. Carboxymethylation after reduction prevents binding to the membrane receptor. The binding properties of the derivative obtained by reduction and aminoethylation of Cys30-Cys34 are very similar to those of native neurotoxin III; the affinity is decreased only by a factor of 5. Binding properties to Toredo membrane of long neurotoxins (Naja haje neurotoxin III) and short neurotoxins (Naje haje toxin I and Naja mossambica toxin I) have been compared. Dissociation constants of receptor-long neurotoxin and receptor-short neurotoxin complexes are very similar (5.7 minus 8.2 times 10(-10) M at pH 7.4, 20degrees. However, the kinetics of complex formation and complex dissociation are quite different. Short neurotoxins associate 6-7 times faster with the toxin receptor and dissociate about 5-9 times faster that long neurotoxins. Acetylation and dansylation of Lys53 and Lys 27 decrease the affinity of long and short toxins for their receptor by a factor of about 200 at pH 7.4, 20 degrees, mainly because of the slower rate of association with the receptor.

show abstract

“…The increased binding affinity of ANS to the trypsin D189S-R 1 -PI complex (Figure 6) and the apparent lability of the disulfide bond network in the complexed proteinase (Figure 7) are also consistent with this view. As to the latter property, a disulfide bond between two loops of the activation domain (Cys 191 -Cys 220 ) may be the "weakest point" of the deformed network of disulfide bridges in the complex, in analogy to the increased accessibility of this bond in trypsinogen as compared to trypsin (Light et al, 1969). Additionally, Stavridi et al (1996) have recently proposed that formation of the R-chymotrypsin-R 1 -antichymotrypsin complex involves displacement or destabilization of the Ile 16 -Gly 25 segment of R-chymotrypsin in order to explain the exposure of sites cleaved by HNE (Val 188 , Ala 158 , and Thr 139 ).…”

Section: Discussionmentioning

confidence: 99%

Effects of Serpin Binding on the Target Proteinase: Global Stabilization, Localized Increased Structural Flexibility, and Conserved Hydrogen Bonding at the Active Site

et al. 1997

View full text Add to dashboard Cite

The binding of human alpha1-proteinase inhibitor to rat trypsin was shown by NMR spectroscopy to raise the pKa' 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 alpha1-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 alpha1-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.

show abstract

Modification of a Single Disulfide Bond in Trypsinogen and the Activation of the Carboxymethyl Derivative

Cited by 29 publications

References 23 publications

Trypsin-pancreatic trypsin inhibitor association. Dynamics of the interaction and role of disulfide bridges

Trypsin-pancreatic trypsin inhibitor association. Dynamics of the interaction and role of disulfide bridges

Structure-function relation in the binding of snake neurotoxins to the Torpedo membrane receptor

Effects of Serpin Binding on the Target Proteinase: Global Stabilization, Localized Increased Structural Flexibility, and Conserved Hydrogen Bonding at the Active Site

Contact Info

Product

Resources

About