Kinetics and Thermodynamics of Interaction Between Soybean Trypsin Inhibitor (Kunitz) and Bovine ß Trypsin

Laskowski, M.; Duran, Ruth W.; Finkenstadt, William R.; Herbert, Sarah; Hixson, Harry F.; Kowalski, David; Luthy, James A.; Mattis, Jeffrey A.; Mckee, Raymond Eugene; Niekamp, Carl W.

doi:10.1515/9783111508146-017

Cited by 5 publications

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“…Inactivation of this inhibitor fraction by maleylation is therefore due to acvlation of the öc-amino group of the X- residue in the reactive site. These findings are in full agreement with the results of LASKOWSKI, Jr. [13] obtained in similar experiments with the modified form of the soybean trypsin inhibitor.…”

Section: Reactive Site Of the Trypsin-specificsupporting

confidence: 92%

“…The reaction mechanism of the modification reactions will not be discussed; it is most elegantly elucidated in the lecture of LASKOWSKI, Jr. et al [13] preceding this paper. The fact that the trypsin-specific inhibitors are obtained in almost completely modified form using the resin method and the trypsin-plasmin inhibitors are unmodified may be explained by different velocity constants of the individual modification steps (see ref.…”

Section: Reactive Site Of the Trypsin-specificmentioning

confidence: 99%

“…The fact that the trypsin-specific inhibitors are obtained in almost completely modified form using the resin method and the trypsin-plasmin inhibitors are unmodified may be explained by different velocity constants of the individual modification steps (see ref. [13]). The reasons for this may be found in differences of the tertiary and primary structure of the reactive sites of these inhibitors.…”

Section: Reactive Site Of the Trypsin-specificmentioning

confidence: 99%

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Protein Proteinase Inhibitors in Male Sex Glands

Fink¹,

Klein²,

Hammer³

et al. 1971

Proceedings of the International Research Conference on Proteinase Inhibitors, Munich, November 4–6, 1970

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Trypsin-specific and trypsin-plasmin inhibitors were isolated from seminal vesicles of guinea pigs. Two different procedures were used: 1. Inhibitor material obtained from perchloric acid extracts was purified by affinity chromatography (using water insoluble trypsin resin) and gradient elution chromatography on Sulfoethyl Sephadex. Mainly two very similar trypsin-specific inhibitors and five somewhat different trypsin-plasmin inhibitors were obtained. (The amino acid compositions are given in Table 3). 2. Also by avoiding the trypsin resin step several inhibitor fractions were obtained which were differing considerably in their amino acid compositions. Inhibitors containing a lysine residue in the reactive site are reversibly inactivated by acylation with maleic anhydride; arginine inhibitors are inactivated by reaction with a butandion-2,3 reagent. In the reactive site of the trypsin-specific inhibitor the sequence Arg-Ile is present. The modified inhibitor (Argile bond is broken) is inactivated by incubation with carboxypeptidase B or reaction with excessive maleic anhydride. The native inhibitor (Arg-Ile bond intact) is converted into the modified form both during contact with the trypsin resin and by incubation with 2.3 mole percent trypsin. From acidic extracts of boar seminal plasma a trypsinplasmin inhibitor was isolated by affinity chromato-This article contains parts of E. FINK, Dissertation,

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Section: Reactive Site Of the Trypsin-specificsupporting

confidence: 92%

Section: Reactive Site Of the Trypsin-specificmentioning

confidence: 99%

Section: Reactive Site Of the Trypsin-specificmentioning

confidence: 99%

See 1 more Smart Citation

Protein Proteinase Inhibitors in Male Sex Glands

Fink¹,

Klein²,

Hammer³

et al. 1971

Proceedings of the International Research Conference on Proteinase Inhibitors, Munich, November 4–6, 1970

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“…Since then, a large number of them have been determined ( , ). The reason for the belief in the suitability of the protein inhibitor−serine proteinase complexes as models for transition state complexes stems in part from the demonstrations in our laboratory ( − ) that standard mechanism protein inhibitors serve the enzymes they inhibit as substrates, albeit very inefficient ones. The mechanisms of enzyme−inhibitor interaction and of enzyme−substrate interaction seem formally identical, differing only in the values of the various on−off rate constants such that in the enzyme−inhibitor system, the lifetime of the complex is very long.…”

mentioning

confidence: 99%

Thermodynamic Criterion for the Conformation of P₁ Residues of Substrates and of Inhibitors in Complexes with Serine Proteinases

Qasim

Ding

et al. 1999

Biochemistry

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Eglin c, turkey ovomucoid third domain, and bovine pancreatic trypsin inhibitor (Kunitz) are all standard mechanism, canonical protein inhibitors of serine proteinases. Each of the three belongs to a different inhibitor family. Therefore, all three have the same canonical conformation in their combining loops but differ in their scaffoldings. Eglin c (Leu45 at P1) binds to chymotrypsin much better than its Ala45 variant (the difference in standard free energy changes on binding is -5.00 kcal/mol). Similarly, turkey ovomucoid third domain (Leu18 at P1) binds to chymotrypsin much better than its Ala18 variant (the difference in standard free energy changes on binding is -4.70 kcal/mol). As these two differences are within the +/-400 cal/mol bandwidth (expected from the experimental error), one can conclude that the system is additive. On the basis that isoenergetic is isostructural, we expect that within both the P1 Ala pair and the P1 Leu pair, the conformation of the inhibitor's P1 side chain and of the enzyme's specificity pocket will be identical. This is confirmed, within the experimental error, by the available X-ray structures of complexes of bovine chymotrypsin Aalpha with eglin c () and with turkey ovomucoid third domain (). A comparison can also be made between the structures of P1 (Lys+)15 of bovine pancreatic trypsin inhibitor (Kunitz) ( and ) and of the P1 (Lys+)18 variant of turkey ovomucoid third domain (), both interacting with chymotrypsin. In this case, the conformation of the side chains is strikingly different. Bovine pancreatic trypsin inhibitor with (Lys+)15 at P1 binds to chymotrypsin more strongly than its Ala15 variant (the difference in standard free energy changes on binding is -1.90 kcal/mol). In contrast, turkey ovomucoid third domain variant with (Lys+)18 at P1 binds to chymotrypsin less strongly than its Ala18 variant (the difference in standard free energies of association is 0.95 kcal/mol). In this case, P1 Lys+ is neither isostructural nor isoenergetic. Thus, a thermodynamic criterion for whether the conformation of a P1 side chain in the complex matches that of an already determined one is at hand. Such a criterion may be useful in reducing the number of required X-ray crystallographic structure determinations. More importantly, the criterion can be applied to situations where direct determination of the structure is extremely difficult. Here, we apply it to determine the conformation of the Lys+ side chain in the transition state complex of a substrate with chymotrypsin. On the basis of kcat/KM measurements, the difference in free energies of activation for Suc-AAPX-pna when X is Lys+ and X is Ala is 1.29 kcal/mol. This is in good agreement with the corresponding difference for turkey ovomucoid third domain variants but in sharp contrast to the bovine pancreatic trypsin inhibitor (Kunitz) data. Therefore, we expect that in the transition state complex of this substrate with chymotrypsin, the P1 Lys+ side chain is deeply inserted into the enzyme's specificity pocket as it is in th...

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The interaction of guanidinated lima bean inhibitor with trypsin

1973

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Kinetics and Thermodynamics of Interaction Between Soybean Trypsin Inhibitor (Kunitz) and Bovine ß Trypsin

Cited by 5 publications

References 0 publications

Protein Proteinase Inhibitors in Male Sex Glands

Protein Proteinase Inhibitors in Male Sex Glands

Thermodynamic Criterion for the Conformation of P₁ Residues of Substrates and of Inhibitors in Complexes with Serine Proteinases

The interaction of guanidinated lima bean inhibitor with trypsin

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Kinetics and Thermodynamics of Interaction Between Soybean Trypsin Inhibitor (Kunitz) and Bovine ß Trypsin

Cited by 5 publications

References 0 publications

Protein Proteinase Inhibitors in Male Sex Glands

Protein Proteinase Inhibitors in Male Sex Glands

Thermodynamic Criterion for the Conformation of P1 Residues of Substrates and of Inhibitors in Complexes with Serine Proteinases

The interaction of guanidinated lima bean inhibitor with trypsin

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Product

Resources

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Thermodynamic Criterion for the Conformation of P₁ Residues of Substrates and of Inhibitors in Complexes with Serine Proteinases