Nuclear magnetic resonance studies of the interaction of trans-cinnamate with .alpha.-chymotrypsin

Gerig, J. T.; Reinheimer, John D.

doi:10.1021/ja00713a037

Cited by 31 publications

(14 citation statements)

References 3 publications

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“…Specifically, resonance peaks of the free ligands are significantly broadened particularly when the off-rate of the ligand binding is fast compared to the NMR time scale and the concentration of the free peptide is higher (> 10-fold) than that of the macromolecule (Dwek, 1973). Protein-induced line broadening of peptide resonances has been utilized in various systems such as chymotrypsin plus peptide substrates (Gerig, 1968;Gerig & Reinheimer, 1970) and neurophysin plus peptide hormones (Balaram et al, 1973) to study the mechanism of the interaction processes.…”

Section: Resultsmentioning

confidence: 99%

High-resolution NMR studies of fibrinogen-like peptides in solution: interaction of thrombin with residues 1-23 of the A.alpha. chain of human fibrinogen

Konishi²,

Frazier³

et al. 1989

Biochemistry

140

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The interaction of the following human fibrinogen-like peptides with bovine thrombin was studied by use of one- and two-dimensional NMR techniques in aqueous solution: Ala(1)-Asp-Ser-Gly-Glu-Gly-Asp-Phe(8)-Leu-Ala-Glu-Gly-Gly-Gly-Val-Arg(16 )- Gly(17)-Pro-Arg(19)-Val(20)-Val-Glu-Arg (F10), residues 1-16 of F10 (fibrinopeptide A), residues 17-23 of F10 (F12), residues 1-20 of F10 (F13), residues 6-20 of F10 with Arg(16) replaced by a Gly residue (F14), and residues 6-19 of F10 with Arg(16) replaced by a Leu residue (F15). At pH 5.3 and 25 degrees C, the Arg(16)-Gly(17) peptide bonds of both peptides F10 and F13 were cleaved instantaneously in the presence of 0.6 mM thrombin, whereas the cleavage of the Arg(19)-Val(20) peptide bonds in peptides F12, F13, and F14 took over 1 h for completion. On the basis of observations of line broadening, fibrinopeptide A was found to bind to thrombin. While resonances from residues Ala(1)-Glu(5) were little affected, binding of fibrinopeptide A to thrombin caused significant line broadening of NH and side-chain proton resonances within residues Asp(7)-Arg(16). There is a chain reversal within residues Asp(7)-Arg(16) such that Phe(8) is brought close to the Arg(16)-Gly(17) peptide bond in the thrombin-peptide complex, as indicated by transferred NOEs between the aromatic ring protons of Phe(8) and the C alpha H protons of Gly(14) and the C gamma H protons of Val(15). A similar chain reversal was obtained in the isolated peptide F10 at a subzero temperature of -8 degrees C. The titration behavior of Asp(7) in peptide F13 does not deviate from that of the reference peptide, N-acetyl-Asp-NHMe at both 25 and -8 degrees C, indicating that no strong interaction exists between Asp(7) and Arg(16) or Arg(19). Peptides with Arg(16) replaced by Gly and Leu, respectively, i.e., F14 and F15, were also found to bind to thrombin but with a different conformation, as indicated by the absence of the long-range NOEs observed with fibrinopeptide A. Residues Asp(7)-Arg(16) constitute an essential structural element in the interaction of thrombin with fibrinogen.

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Section: Resultsmentioning

confidence: 99%

High-resolution NMR studies of fibrinogen-like peptides in solution: interaction of thrombin with residues 1-23 of the A.alpha. chain of human fibrinogen

Konishi²,

Frazier³

et al. 1989

Biochemistry

140

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“…Typically the concentrations of enzyme, dye, and inhibitor were 2-6 X 10-5, 9 X 10-5, and 0.4-1 X 10-3, M, respectively. 6 Nonlinear semilog plots observed after ca. 2 half-lives. "…”

Section: Resultsmentioning

confidence: 99%

Kinetics of inactivation of .alpha.-chymotrypsin with substituted benzenesulfonyl fluorides

Gerig¹,

Roe²

1974

J. Am. Chem. Soc.

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The dissociation constants for complexes of chymotrypsin Aa with a series of (V-acyl-and A-peptidyl-2phenylethylamine competitive inhibitors were measured in a 9.5 % ethanol, 0.10 M CaCls solution at pH 7.80 at 25°. Individual subsite binding free energies for the extended substrate binding site in chymotrypsin were calculated. The free energies of binding in this series of vV-acyl-and A-peptidyl-2-phenylethylamines become more negative with increasing number of interactions with enzyme as predicted from the crystallographic model. The major contributors to the binding free energy are hydrophobic interactions, the Si-Pi tosyl pocket-phenylethyl group interaction(2 kcal/mol) and the interaction of an aliphatic side chain of a P2 residue with Ile-99 in the S2 binding subsite of the enzyme. The individual subsite binding free energies are not additive. Comparison of the dissociation constants of Á-acyl-and X-peptidyl-2-phenylethylamines with the Xm values for the corresponding substrates indicates that the inhibitors are more tightly bound. This is consistent with the hypothesis that serine proteases place the scissionable peptide bond of a substrate on a stereoelectronic "rack" favoring formation of a tetrahedral intermediate. RecentX-ray studies on chymotrypsin A7 crystals .

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“…Information about the active sites is often inferred from the effects that the enzyme has on the chemical shifts (9,10), and line-widths (11), as well as on relaxation times (12) of specific resonances of the ligand.…”

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confidence: 99%

Conformations of Diphosphopyridine Coenzymes upon Binding to Dehydrogenases

Lee¹,

Eichner²,

Kaplan³

1973

Proc. Natl. Acad. Sci. U.S.A.

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The binding of oxidized as well as reduced coenzyme to some dehydrogenases has been studied under different concentration ratios and temperatures by nuclear magnetic resonance spectroscopy. A significant difference in the spectral behavior between DPN+ and DPNH upon binding is interpreted in terms of fast and slow on-off rates relative to the nuclear magnetic resonance time scale in the binding of these two coenzymes. Significant downfield shifts of DPN+ were observed upon binding, comparable in magnitude to those expected upon opening (destacking) of the coenzymes in the case of chicken-muscle and lobster-tail lactate dehydrogenase (EC 1.1.1.27) and yeast alcohol dehydrogenase (EC 1.1.1.1). A preliminary survey of several other dehydrogenases is consistent with these findings. In the case of 3-phosphoglyceraldehyde dehydrogenase, there is a possibility that the coenzyme exists in the folded form.Nuclear magnetic resonance spectroscopy (NMR) is now extensively applied to probe the active sites of enzymes in solution (1, 2). Most of these studies have been concerned with reversible enzyme-inhibitor or substrate interactions (3-8).Information about the active sites is often inferred from the effects that the enzyme has on the chemical shifts (9, 10), and line-widths (11), as well as on relaxation times (12) of specific resonances of the ligand.It has been shown that in neutral aqueous solution the pyridine coenzymes, DPN+ and DPNH, exist in rapid equilibrium between open and folded conformations (13-15). Investigations regarding the actual conformations of these coenzymes in the active sites of dehydrogenases showed discrepancies depending on the methodology used. The fluorescence energy-transfer study of Velick indicates that the reduced coenzyme appears to exist in an open conformation upon binding to most dehydrogenases; 3-phosphoglyceraldehyde dehydrogenase being the only exception (16,17). From a 5A x-ray map, Adams et al. (18) 1593 the reduced coenzyme exists in an intramolecular folded conformation in the active site of this enzyme (19). The observation of the upfield shift of both H8 and H2 protons on the adenine moiety of DPNH in the presence of this enzyme led to the above conclusions.We have recently studied again the binding of pyridine coenzymes to various dehydrogenases under various experimental conditions. From the results of our detailed study, we have been able to ascertain the conformation of coenzymes when bound to dehydrogenases in solution. MATERIALS AND METHODSAlcohol dehydrogenase from yeast (YADH) was purchased from both Calbiochem, Calif. and Sigma Chemical Corp., Mo. Lactate dehydrogenase from chicken muscle (M4LDH) and chicken heart (H4LDH) were prepared in our laboratory by Mr. Francis Stolzenbach (20). Lactate dehydrogenase from lobster tail was isolated and purified as reported elsewhere (21,22). The specific activities of dehydrogenases used in this study were measured before each experiment.

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Nuclear magnetic resonance studies of the interaction of trans-cinnamate with .alpha.-chymotrypsin

Cited by 31 publications

References 3 publications

High-resolution NMR studies of fibrinogen-like peptides in solution: interaction of thrombin with residues 1-23 of the A.alpha. chain of human fibrinogen

High-resolution NMR studies of fibrinogen-like peptides in solution: interaction of thrombin with residues 1-23 of the A.alpha. chain of human fibrinogen

Kinetics of inactivation of .alpha.-chymotrypsin with substituted benzenesulfonyl fluorides

Conformations of Diphosphopyridine Coenzymes upon Binding to Dehydrogenases

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