Kinetics and Thermodynamics of the Interaction of Proteinases with Protein Inhibitors

Finkenstadt, William R.; Hamid, Muhammad Akhter; Mattis, Jeffrey A.; Schrode, James; Sealock, Robert; Wang, D.; LaskowskiJr., M.

doi:10.1007/978-3-642-87966-1_45

Cited by 53 publications

(35 citation statements)

References 32 publications

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“…3 shows that a good fit was obtained with K, = 6.5 x M. With this equilibrium constant and with the value of the apparent rate constant of the fast phase kapp = 0.35 s-' (see Fig. 1) the rate constant k , in mechanism (1) was calculated according to (3) and equalled k , = 4 x lo3 M-' S -l .…”

Section: Evidence J O R An Intermediatementioning

confidence: 85%

“…The rate constants and equilibrium constants at pH 7.5 are summarized in Table 1, which also contains the corresponding values for P-trypsin. The rate constant kcould not be measured because C dissociates faster into E + I than towards E + I* [3]. k -, was therefore (Table 1).…”

Section: Discussionmentioning

confidence: 99%

“…After formation of C from I*-OMe the methyl group is removed from Lys-15 but not from other groups which are possibly esterified. Dissociation of C, which proceeds toward the virgin side [3], was induced by addition of 4-nitrophenyl-N2-acetyl-N'-benzylcarbazate [12]. Depending upon pH the following rate constants of dissociation were observed 4.6 x s-' at pH5, 2.6 s -' at pH6.…”

Section: Rrurtion Of A-chymotrypsin With I*-omtjmentioning

confidence: 99%

“…This bond is catalytically split by the enzyme. The reaction in which modified inhibitor I* is formed leads to an equilibrium [I*]/[I] = Khrd near unity for many inhibitors [3] at neutral pH. In the case of trypsin kallikrein inhibitor the slow cleavage of the reactive site peptide 'bond remained first undetected [4] but recently Tschesche and Kupfer found equilibrium constants Khrd = 0.3 -0.38 at pH 5-7.5 [5,6].…”

mentioning

confidence: 99%

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Kinetics of the Interaction of α‐Chymotrypsin with Trypsin Kallikrein Inhibitor (Kunitz) in Which the Reactive‐Site Peptide Bond Lys‐15–Ala‐16 is Split

Quast¹,

Engel²,

Steffen³

et al. 1978

European Journal of Biochemistry

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Modified trypsin kallikrein inhibitor (I*), with the reactive‐site peptide bond Lys‐15–Ala‐16 split, reacts with α‐chymotrypsin (E) via an intermediate X to the stable tetrahedral complex C: E + I*⇌ X → C. Formation of X constitutes a fast pre‐equilibrium (equilibrium constant Kx= 7 × 10−5 M. association rate constant kx= 4 × 103 M−1 s−1) to the slow reaction X → C (rate constant kc= 2 × 10−3 s−1), all values at pH 7.5. No intermediate X is observed when α‐chymotrypsin reacts with I*‐OMe in which the carboxyl group of Lys‐15 is esterified by methanol. This observation as well as the different pH dependence of the overall association rate constants in the case of I* and I*‐OMe indicate that formation of X precedes formation of the acyl enzyme in the catalytic pathway. The data are compared to the similar results obtained with β‐trypsin and I* or I*‐OMe.

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Section: Evidence J O R An Intermediatementioning

confidence: 85%

Section: Discussionmentioning

confidence: 99%

Section: Rrurtion Of A-chymotrypsin With I*-omtjmentioning

confidence: 99%

mentioning

confidence: 99%

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Kinetics of the Interaction of α‐Chymotrypsin with Trypsin Kallikrein Inhibitor (Kunitz) in Which the Reactive‐Site Peptide Bond Lys‐15–Ala‐16 is Split

Quast¹,

Engel²,

Steffen³

et al. 1978

European Journal of Biochemistry

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“…For example, the complexes of trypsin with soy bean trypsin inhibitor (47) and with the squash inhibitors(59) from Cucurbita maxima, as well the complex of subtilisin with CI2 (60;5) all have dissociation constants in the range of 10 −12 to 10 −11 M and half times for hydrolysis on the order of one to ten days. The functionality of Y35G BPTI persists despite the extensive motions of the trypsin-binding region in the free inhibitor, and likely depends on rigidity imparted to the loops as a result of the intermolecular interactions formed upon binding.…”

Section: Functional Consequences Of the Y35g Replacementmentioning

confidence: 99%

Rigidification of a Flexible Protease Inhibitor Variant upon Binding to Trypsin

Hanson

Domek

Horvath

et al. 2007

Journal of Molecular Biology

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The Tyr35→Gly replacement in bovine pancreatic trypsin inhibitor (BPTI) has previously been shown to dramatically enhance the flexibility of the trypsin-binding region of the free inhibitor and to destabilize the interaction with the protease by about 3 kcal/mol. The effects of this replacement on the enzyme-inhibitor interaction were further studied here by x-ray crystallography and isothermal titration calorimetry. The co-crystal structure of Y35G BPTI bound to trypsin was determined using 1.65 Å resolution x-ray diffraction data collected from cryopreserved crystals, and a new structure of the complex with wild-type BPTI under the same conditions was determined using 1.62 Å data. These structures reveal that, in contrast to the free protein, Y35G BPTI adopts a conformation nearly identical to that of the wild-type protein, with a water-filled cavity in place of the missing Tyr side chain. The crystallographic temperature factors for the two complexes indicate that the mutant inhibitor is nearly as rigid as the wild-type protein when bound to trypsin. Calorimetric measurements show that the change in enthalpy upon dissociation of the complex is 2.5 kcal/mol less favorable for the complex containing Y35G BPTI than for the complex with the wild-type inhibitor. Thus, the destabilization of the complex resulting from the Y35G replacement is due to a more favorable change in entropy upon dissociation. The heat capacity changes for dissociation of the mutant and wild-type complexes were very similar, suggesting that the entropic effects probably do not arise from solvation effects, but are more likely due to an increase in protein conformational entropy upon dissociation of the mutant inhibitor. These results define the biophysical role of a highly conserved core residue located outside of a protein-binding interface, demonstrating that Tyr35 has little impact on the trypsin-bound BPTI structure and acts primarily to define the structure of the free protein so as to maximize binding affinity. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.Abbreviations used: BPTI, bovine pancreatic trypsin inhibitor; Amino acid replacements are indicated by the wild-type residue type (using the one letter code for the 20 standard amino acid residues), followed by the residue number and the mutant residue type; Elsewhere, amino acid residue types are indicated by the standard 3-letter code. IAS, iso-aspartate; HEPES, N -2-Hydroxyethylpiperazine-N′-2-ethanesulfonic acid; Tris, tris(hydroxymethyl)-aminomethane; NMR, nuclear magnetic resonance.

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Proteinase‐Catalyzed Synthesis of Peptide Bonds

Fruton

1982

Advances in Enzymology - And Related Areas of Molecular Biology

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Kinetics and Thermodynamics of the Interaction of Proteinases with Protein Inhibitors

Cited by 53 publications

References 32 publications

Kinetics of the Interaction of α‐Chymotrypsin with Trypsin Kallikrein Inhibitor (Kunitz) in Which the Reactive‐Site Peptide Bond Lys‐15–Ala‐16 is Split

Kinetics of the Interaction of α‐Chymotrypsin with Trypsin Kallikrein Inhibitor (Kunitz) in Which the Reactive‐Site Peptide Bond Lys‐15–Ala‐16 is Split

Rigidification of a Flexible Protease Inhibitor Variant upon Binding to Trypsin

Proteinase‐Catalyzed Synthesis of Peptide Bonds

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