Motion at the active site of [(4-fluorophenyl)sulfonyl]chymotrypsin

Ando, Masashi; Gerig, J. T.; Luk, Kong

doi:10.1021/bi00365a008

Cited by 9 publications

(4 citation statements)

References 29 publications

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“…As is apparent from this figure, each of the fluorine resonances which appear in the 19F NMR spectrum of the 5-fluorotryptophan-labeled enzyme (Luck and Falke, 1991a) is now doubled. The observed isotope-shift value of-0.58 ppm is approximately double the value of-0.262 ppm, which has been reported for each ortho-deuterium substitution in the [3,5-2H2]-4-fluorobenzenesulfonyl fluoride (Ando et al, 1986), and is also consistent with the 7-deuterium isotope shifts observed in fluoroethanes (Osten et al, 1985). The resonances due to FTrp 133 now appear as three separate peaks due to the combined effects of the conformational shift (Av = 0.65 ppm) and the isotope shift, with the resonance furthest upfield overlapping one of the FTrp 284 resonances.…”

Section: Resultsmentioning

confidence: 47%

19F NMR relaxation studies on 5-fluorotryptophan- and tetradeutero-5-fluorotryptophan-labeled E. coli glucose/galactose receptor

et al. 1996

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19F NMR relaxation studies have been carried out on a fluorotryptophan-labeled E. coli periplasmic glucose/galactose receptor (GGR). The protein was derived from E. coli grown on a medium containing a 50:50 mixture of 5-fluorotryptophan and [2,4,6,7-2H4]-5-fluorotryptophan. As a result of the large gamma-isotope shift, the two labels give rise to separate resonances, allowing relaxation contributions of the substituted indole protons to be selectively monitored. Spin-lattice relaxation rates were determined at field strengths of 11.75 T and 8.5 T, and the results were analyzed using a model-free formalism. In order to evaluate the contributions of chemical shift anisotropy to the observed relaxation parameters, solid-state NMR studies were performed on [2,4,6,7-2H4]-5-fluorotryptophan. Analysis of the observed 19F powder pattern lineshape resulted in anisotropy and asymmetry parameters of delta sigma = -93.5 ppm and eta = 0.24. Theoretical analyses of the relaxation parameters are consistent with internal motion of the fluorotryptophan residues characterized by order parameters S2 of approximately 1, and by correlation times for internal motion approximately 10(-11)s. Simultaneous least squares fitting of the spin-lattice relaxation and line-width data with tau i set at 10 ps yielded a molecular correlation time of 20 ns for the glucose-complexed GGR, and a mean order parameter S2 = 0.89 for fluorotryptophan residues 183, 127, 133, and 195. By contrast, the calculated order parameter for FTrp284, located on the surface of the protein, was 0.77. Significant differences among the spin-lattice relaxation rates of the five fluorotryptophan residues of glucose-complexed GGR were also observed, with the order of relaxation rates given by: R1F183 > R1F127 approximately R1F133 approximately R1F195 > R1F284. Although such differences may reflect motional variations among these residues, the effects are largely predicted by differences in the distribution of nearby hydrogen nuclei, derived from crystal structure data. In the absence of glucose, spin-lattice relaxation rates for fluorotryptophan residues 183, 127, 133, and 195 were found to decrease by a mean of 13%, while the value for residue 284 exhibits an increase of similar magnitude relative to the liganded molecule. These changes are interpreted in terms of a slower overall correlation time for molecular motion, as well as a change in the internal mobility of FTrp284, located in the hinge region of the receptor.

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Section: Resultsmentioning

confidence: 47%

19F NMR relaxation studies on 5-fluorotryptophan- and tetradeutero-5-fluorotryptophan-labeled E. coli glucose/galactose receptor

et al. 1996

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“…Fluorescence correlation spectroscopy indicates that the rotational correlation time for CA I should be 12-15 ns (Kask et al, 1987). If one assumes a standard geometry for the nuclei attached to the aromatic ring of I and assumes that a proton of the enzyme comes within van der Waals contact of the fluorine nucleus of the inhibitor, the relaxation of the fluorine nucleus of a bound inhibitor molecule should be similar to that deduced for [(4-fluorophenyl)sulfonyl]chymotrypsin (Ando et al, 1986) where the fluorine Tx and NOE values and the rotational correlation time are all similar to those found for carbonic anhydrase. In the case of the chymotrypsin derivative, it was concluded that rotation of the aromatic ring holding the fluorine atom was significantly hindered, being characterized by a correlation time slightly smaller than that for overall tumbling of the protein.…”

Section: Discussionmentioning

confidence: 93%

NMR studies of carbonic anhydrase-4-fluorobenzenesulfonamide complexes

Dugad¹,

Gerig²

1988

Biochemistry

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Binding of 4-fluorobenzenesulfonamide to human carbonic anhydrases I and II has been studied by proton, fluorine, and nitrogen-15 nuclear magnetic resonance spectroscopy. All three types of experiments provide evidence that the stoichiometry of the interaction of this inhibitor with both enzymes is 2 mol of inhibitor bound per mole of enzyme. Observations which suggest that the bound forms are involved in an exchange process that is rapid at room temperature but slower at 2 degrees C are described. Nitrogen-15 shift data show that the bound inhibitors are present at the active site as anions. The proton experiments indicate appreciable reorganization of the tertiary structure of the protein upon binding. Saturation-transfer experiments to determine the rate of dissociation of the inhibitor-enzyme complex lead to the conclusion that the dissociation process is more complicated than a simple free-bound equilibrium.

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“…It has been frequently observed that fluorine linewidths in protein systems are substantially broader than would be anticipated from spin-lattice relaxation behavior (for examples see Kimber et ah (1977), Post et al (1984), and Ando, Gerig and Luk (1986)) and we ignored the linewidth data in attempting to analyze the dynamics of these systems. Relaxation of aromatic fluorine of IV in the presence of the enzyme was examined by setting up a program to compute T i relaxation behavior and time dependence of tgF{tH} NOE formation using the model system described above.…”

Section: Resultsmentioning

confidence: 99%

Structure and dynamics of α-chymotrypsin-N-trifluoroacetyl-4-fluorophenylalanine complexes

Jacobson

Gerig

1991

J Biomol NMR

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Fluorine NMR lineshape, relaxation and Overhauser effect data collected at 282 and 470 MHz have been used to obtain information about the nature of complexes formed between N-trifluoroacetyl-4-fluorophenylalanine and the enzyme chymotrypsin. Systems involving both enantiomers have been examined as well as derivatives of these in which the aromatic ring hydrogens have been replaced by deuterium. The enzyme-induced fluorine chemical shift effects and the dynamics of molecular motions of the fluorophenyl ring at the respective binding sites appear to be similar in both complexes and, where comparable, the results are in agreement with data obtained at lower frequencies that have been reported by other workers. The dynamics of the fluoroaromatic ring in these complexes are significantly different from those observed in a closely related acylated enzyme.

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Motion at the active site of [(4-fluorophenyl)sulfonyl]chymotrypsin

Cited by 9 publications

References 29 publications

19F NMR relaxation studies on 5-fluorotryptophan- and tetradeutero-5-fluorotryptophan-labeled E. coli glucose/galactose receptor

19F NMR relaxation studies on 5-fluorotryptophan- and tetradeutero-5-fluorotryptophan-labeled E. coli glucose/galactose receptor

NMR studies of carbonic anhydrase-4-fluorobenzenesulfonamide complexes

Structure and dynamics of α-chymotrypsin-N-trifluoroacetyl-4-fluorophenylalanine complexes

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