[32] Differential spectrofluorometry

Fasman, Gerald D.; Bablouzian, B.

doi:10.1016/s0076-6879(73)27035-0

Cited by 6 publications

(5 citation statements)

References 13 publications

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“…Keywords: C À F bond activation • conformational analysis • docking simulations • enzyme catalysis • selectivity the formation of an ester intermediate, which is subsequently hydrolyzed by the nucleophilic attack of a water molecule mediated by His271. This mechanism is consistent with 18 O labeling and mutational studies. [15] In our previous study, [16] we carried out quantum mechanical/molecular mechanical (QM/MM) calculations to obtain better information about structural and catalytic interactions in the active site using the crystal structure.…”

Section: Introductionsupporting

confidence: 89%

“…The difference spectrum of “WT” minus “W150F” represents the fluorescence of Trp150, which shows a maximum at about 350 nm (Figure 2 B). This result indicates that Trp150 is located in a highly polar environment 18, 19. The active site of the enzyme, where Trp150 is located, is probably readily accessible to the solvent.…”

Section: Resultsmentioning

confidence: 88%

“…This result indicates that Trp150 is located in a highly polar environment. [18,19] The active site of the enzyme, where Trp150 is located, is probably readily accessible to the solvent. When fluoroacetate was added to the wild-type enzyme, the fluorescence spectrum became similar to that of the Trp150Phe mutant; this suggests that the fluorescence of Trp150 was almost completely quenched upon binding of fluoroacetate to the enzyme.…”

Section: Resultsmentioning

confidence: 99%

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Substrate Specificity of Fluoroacetate Dehalogenase: An Insight from Crystallographic Analysis, Fluorescence Spectroscopy, and Theoretical Computations

Nakayama

Kamachi

Jitsumori

et al. 2012

Chemistry A European J

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The high substrate specificity of fluoroacetate dehalogenase was explored by using crystallographic analysis, fluorescence spectroscopy, and theoretical computations. A crystal structure for the Asp104Ala mutant of the enzyme from Burkholderia sp. FA1 complexed with fluoroacetate was determined at 1.2 Å resolution. The orientation and conformation of bound fluoroacetate is different from those in the crystal structure of the corresponding Asp110Asn mutant of the enzyme from Rhodopseudomonas palustris CGA009 reported recently (J. Am. Chem. Soc. 2011, 133, 7461). The fluorescence of the tryptophan residues of the wild‐type and Trp150Phe mutant enzymes from Burkholderia sp. FA1 incubated with fluoroacetate and chloroacetate was measured to gain information on the environment of the tryptophan residues. The environments of the tryptophan residues were found to be different between the fluoroacetate‐ and chloroacetate‐bound enzymes; this would come from different binding modes of these two substrates in the active site. Docking simulations and QM/MM optimizations were performed to predict favorable conformations and orientations of the substrates. The F atom of the substrate is oriented toward Arg108 in the most stable enzyme–fluoroacetate complex. This is a stable but unreactive conformation, in which the small OCF angle is not suitable for the SN2 displacement of the F− ion. The cleavage of the CF bond is initiated by the conformational change of the substrate to a near attack conformation (NAC) in the active site. The second lowest energy conformation is appropriate for NAC; the CO distance and the OCF angle are reasonable for the SN2 reaction. The activation energy is greatly reduced in this conformation because of three hydrogen bonds between the leaving F atom and surrounding amino acid residues. Chloroacetate cannot reach the reactive conformation, due to the longer CCl bond; this results in an increase of the activation energy despite the weaker CCl bond.

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Section: Introductionsupporting

confidence: 89%

Section: Resultsmentioning

confidence: 88%

Section: Resultsmentioning

confidence: 99%

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Substrate Specificity of Fluoroacetate Dehalogenase: An Insight from Crystallographic Analysis, Fluorescence Spectroscopy, and Theoretical Computations

Nakayama

Kamachi

Jitsumori

et al. 2012

Chemistry A European J

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“…The reagent has been found to possess striking selectivity in modifying tryptophan residues at acid pH, although the (116) sulfhydryl group of cysteine also reacts at a much slower rate. At alkaline pH, cysteine and tyrosine react at about the same rate as tryptophan.…”

Section: Chemistry Of the Reactionmentioning

confidence: 99%

“…Information concerning the environment and location of tryptophan side chains and the changes in environment of these residues due to structural transformations can be gained by ultraviolet difference spectroscopy and fluorescence measurements. However, since these two techniques are weil known to biochemists and have been reviewed extensively (55,101,116,172,410,411), it was decided to Iimit the following presentation to other modern spectroscopic tools. Among those particularly worthy of mention appear to be circular dichroism, magnetic circular dichroism, circular polarization of fluorescence, nuclear magnetic resonance eH, 13 C, 14 N) and X-ray diffraction.…”

Section: Structure Determinationmentioning

confidence: 99%