NMR studies of the α-chymotrypsin-(R)-1-acetamido-2-(4-fluorophenyl)ethane-1-boronic acid complex

Sylvia, L.A.; Gerig, J. T.

doi:10.1016/0167-4838(93)90169-r

Cited by 8 publications

(8 citation statements)

References 52 publications

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“…It then follows that the two peaks originating from bound species in the 3TFMB spectra (Figures 2C and 4A) and 2TFMC (Figure 7B) each arise from two different binding modes within the same R 3 binding site, corresponding to two different chemical shift environments for the CF 3 group. Similar behavior has been reported for the binding of fluorinated ligands to other protein sites (23,26,32,35).…”

Section: Discussionsupporting

confidence: 82%

“…Since two resonances are observed when 3TFMB or 2TFMC bind to T 3 R 3 (Figures 4 and 7A,B), we conclude that the flipping rate is slowed by the steric clashing of the CF 3 group with the walls of the site, giving two peaks. Gerig's 19 F NMR work provides ample precedent for slow ring flipping rates in protein ligand complexes (23,26,32,35).…”

Section: Discussionmentioning

confidence: 99%

“…These studies establish that trifluoromethyl-substituted benzoates and cinnamates are sensitive probes of insulin conformation in solution and are capable of distinguishing between the T 3 R 3 and R 6 states of the Zn(II)-and Co(II)-substituted insulin hexamers. As will be shown, this sensitivity to conformation state in the insulin system arises from the following factors: (1) the sensitivity of 19 F chemical shifts to small changes in environment is relatively high (for example in comparison to 1 H chemical shifts) (23)(24)(25)(26)(27), (2) the formation of bound states that are in slow exchange with the free ligand on the NMR time scale, and (3) the simplicity of the 19 F NMR spectra of complexes obtained with ligands containing CF 3 -substituents. As will be shown in this study, we have succeeded for the first time in detecting and quantifying the number and distribution of allosteric conformation states present in the T-to R-state transitions of the insulin hexamer.…”

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confidence: 99%

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Insulin Allosteric Behavior: Detection, Identification, and Quantification of Allosteric States via ¹⁹F NMR

et al. 2005

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The insulin hexamer is an allosteric protein widely used in formulations for the treatment of diabetes. The hexamer exhibits positive and negative cooperativity and apparent half-site binding activity, reflecting the interconversion of three allosteric states, designated as T6, T3R3, and R6. The hexamer contains two symmetry-related Zn2+ located 16 A apart on the 3-fold symmetry axis. In the transition of T3 units to R3 units, Zn2+ switches from an octahedral Zn2+ N3O3 complex (N is HisB10, O is H2O) to a distorted tetrahedral Zn2+ N3L complex (L is a monovalent anion). Hence, monovalent anions are allosteric ligands that stabilize R3 units of T3R3 and R6. Herein, we exploit the high sensitivity of 19F NMR chemical shifts and fluorinated carboxylates to reveal subtle differences in the anion-binding sites of T3R3 and R6. We show that the chemical shifts of 4- and 3-trifluoromethylbenzoate and 4- and 2-trifluoromethylcinnamate give bound resonances that distinguish between T3R3 and R6. 3-Trifluoromethylbenzoate and 2-trifluoromethylcinnamate also were shown to bind to the R3 units of T3R3 and R6 in two alternative, slowly interconverting modes with different microenvironments for the CF3 groups. Line width analysis shows that ligand off rates are slower by 1/10(3) than the diffusion limit, indicating a rate-limiting protein conformational transition. These studies confirm that the Seydoux, Malhotra, and Bernhard allosteric model (Bloom, C. R., Choi, W. E., Brzovic, P. S., Ha, J. J., Huang, S. T., Kaarsholm, N. C., and Dunn, M. F. (1995). J. Mol. Biol. 245, 324-330), provides a robust description of the insulin hexamer.

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Section: Discussionsupporting

confidence: 82%

Section: Discussionmentioning

confidence: 99%

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confidence: 99%

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Insulin Allosteric Behavior: Detection, Identification, and Quantification of Allosteric States via ¹⁹F NMR

et al. 2005

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“…Theories of enzyme catalysis invoking three point attachment for stereoselectivity (1), ground-state destabilization (2), or transition state stabilization (3) imply that substrates are firmly bound to enzyme active sites. However, examination of motions of substrates or inhibitors bound to enzyme active sites show the bound molecules can have significant mobility (4)(5)(6)(7)(8)(9)(10). The relationship between the dynamics of bound substrates and the rate of the chemical step of the catalyzed reaction is not well understood.…”

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confidence: 99%

Mobility of Fluorobenzyl Alcohols Bound to Liver Alcohol Dehydrogenases as Determined by NMR and X-ray Crystallographic Studies^,

Rubach

Plapp

2002

Biochemistry

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The relationship between substrate mobility and catalysis was studied with wild-type and Phe93Ala (F93A) horse liver alcohol dehydrogenase (ADH). Wild-type ADH binds 2,3,4,5,6-pentafluorobenzyl alcohol in one position as shown by X-ray results, and (19)F NMR shows five resonances for the fluorines of the bound alcohol. The two meta-fluorines exchange positions with a rate constant of about 4 s(-1), indicating that mobility (ring flipping) of the benzyl alcohol is relatively restricted. The wild-type enzyme binds 2,3-difluorobenzyl alcohol in two alternative conformations that are related by a ring flip and a small translation of the fluorinated benzene ring, and the (19)F NMR spectrum shows three resonances for the two bound fluorines, consistent with the two orientations. Phe-93 interacts with the bound benzyl alcohols, and the F93A substitution decreases the rate constants for hydride transfer for benzyl alcohol oxidation and benzaldehyde reduction by 7.4- and 130-fold, respectively. The structure of F93A ADH crystallized with NAD(+) and 2,3,4,5,6-pentafluorobenzyl alcohol is similar to the structure of the wild-type enzyme complex except that the pentafluorobenzyl alcohol is not found in one position. The (19)F NMR spectrum of the F93A ADH-NAD(+)-pentafluorobenzyl alcohol complex shows three resonances for the bound fluorines. Line shape analysis of the spectrum suggests the bound pentafluorobenzyl ring undergoes rapid ring-flipping at about 20 000 s(-1). The F93A substitution greatly increases the mobility of the benzyl alcohol but modestly and differentially decreases the probability that the substrate is preorganized for hydride transfer.

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“…h i b i t e d .downfield compared to their resonance values in dUrd (Urd) as a consequence of the electron-withdrawing effects of the F nucleus bound to the adjacent carbon(Sylvia & Gerig, 1993). Smaller differences in chemical shift are observed for the H8 resonances of dA residues in dA-FdUrd (A-FUrd) base pairs that are shifted downfield by about 0.04 ppm compared to the resonance values in dA-dUrd (A-Urd) base pairs, indicating that the electron-withdrawing inductive effect of 19 F is transmitted through hydrogen bonds as well as through the π-electron system of the aromatic base.19 NMR spectra show one resonance from the single FUrd in [FUR] and [UFR] and two resonances from each of the two FUrd nucleosides in [FFR] (Figure2).…”

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confidence: 99%

Solution Structures of 5-Fluorouracil-Substituted DNA and RNA Decamer Duplexes

1996

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The structures in solution of eight oligonucleotide duplexes each containing either zero, one, or two 5-fluorodeoxyuridine (FdUrd) or 5-fluorouridine (FUrd) nucleosides were determined by the combined use of NMR spectroscopy, restrained molecular dynamics, and full relaxation matrix refinement to determine how FdUrd and FUrd substitution affects the structure of duplex DNA and RNA and to establish whether structural differences due to FdUrd and FUrd substitution in nucleic acids may be responsible, in part, for the biological effects of the anticancer drug 5-fluorouracil (FUra). The nucleic acid directed effects of FUra include induction of single-strand breaks in duplex DNA and altered processing of pre-mRNA and rRNA. Four self-complementary oligodeoxyribonucleotide sequences were prepared and studied as duplexes in aqueous solution: (5' dGCGAAUUCGC)2, (5' dGCGAAUFCGC)2, (5' dGCGAAFUCGC)2, and (5' dGCGAAFFCGC)2. The corresponding oligoribonucleotide sequences (5' rGCGAAUUCGC)2, (5' rGCGAAUFCGC)2, (5' rGCGAAFUCGC)2, and (5' rGCGAAFFCGC)2 were also prepared and studied. The helical parameters for the structures of these eight duplexes were analyzed to determine how substitution of FdUrd and FUrd affects the three-dimensional structures of duplex DNA and RNA. FdUrd substitution affects the base roll angle at the site of FdUrd substitution, causing the helical axis of FdUrd-substituted DNA duplexes to be bent compared to the nonsubstituted duplex. A-FUrd base pairs show substantial RMS deviations from A-Urd base pairs in all three of the RNA duplexes substituted with FUrd. Bending of the helical axis due to FdUrd substitution may contribute to the occurrence of single-strand breaks in duplex DNA while the altered structures of A-FUrd base pairs may affect RNA-RNA and RNA-protein recognition.

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NMR studies of the α-chymotrypsin-(R)-1-acetamido-2-(4-fluorophenyl)ethane-1-boronic acid complex

Cited by 8 publications

References 52 publications

Insulin Allosteric Behavior: Detection, Identification, and Quantification of Allosteric States via ¹⁹F NMR

Insulin Allosteric Behavior: Detection, Identification, and Quantification of Allosteric States via ¹⁹F NMR

Mobility of Fluorobenzyl Alcohols Bound to Liver Alcohol Dehydrogenases as Determined by NMR and X-ray Crystallographic Studies^,

Solution Structures of 5-Fluorouracil-Substituted DNA and RNA Decamer Duplexes

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NMR studies of the α-chymotrypsin-(R)-1-acetamido-2-(4-fluorophenyl)ethane-1-boronic acid complex

Cited by 8 publications

References 52 publications

Insulin Allosteric Behavior: Detection, Identification, and Quantification of Allosteric States via 19F NMR

Insulin Allosteric Behavior: Detection, Identification, and Quantification of Allosteric States via 19F NMR

Mobility of Fluorobenzyl Alcohols Bound to Liver Alcohol Dehydrogenases as Determined by NMR and X-ray Crystallographic Studies,

Solution Structures of 5-Fluorouracil-Substituted DNA and RNA Decamer Duplexes

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Insulin Allosteric Behavior: Detection, Identification, and Quantification of Allosteric States via ¹⁹F NMR

Insulin Allosteric Behavior: Detection, Identification, and Quantification of Allosteric States via ¹⁹F NMR

Mobility of Fluorobenzyl Alcohols Bound to Liver Alcohol Dehydrogenases as Determined by NMR and X-ray Crystallographic Studies^,