Tryptophan fluorescence quenching by enzyme inhibitors as a tool for enzyme active site structure investigation

Epoxide hydrolase

Evgenia G. Matveeva, Christophe Morisseau, Marvin H. Goodrow, Chris Mullin, Bruce D. Hammock

Research output: Contribution to journalArticle

14 Citations (Scopus)

Abstract

We present the strong fluorescence effect, a new 392 nm emission peak appearing after binding of a naphtolurea inhibitor XIIa to the enzyme epoxide hydrolase (EH), along with the quenching of the EH tryptophan fluorescence. We have studied the quenching of the 392-nm peak (attributed to XIIa bound inside the active center of the enzyme) of the mixture EH+XIIa by various strong transparent inhibitors (competing with XIIa for binding to EH), and measured the corresponding values of the Stern-Volmer constants, K(mix)SV. Strong EH inhibitors demonstrate different replacement behavior which can be used to distinguish them. We further demonstrate a novel fluorescent assay which allows to distinguish highly potent inhibitors and to visualize the strongest among them. We generated our assay calibration curve based on the quenching data, by plotting quenching strength K(mix)SV versus inhibiting strength, IC50 values. We used moderate inhibitors for the assay plot generation. We then applied this curve to determine IC50 values for several highly potent inhibitors, with IC50 values at the limit of the IC50 detection sensitivity by colorimetric enzyme assay. IC 50 values determined from our quenching assay show correlation with IC50 values determined in the literature by more sensitive radioactive-based assay and allow differentiating the inhibitors potency in this group. To our knowledge, this is the first inhibitor assay of such kind. Chemical inhibition of EH is an important technology in the treatment of various cardiovascular diseases, therefore, this tool may play a crucial role in discovering new inhibitor structures for therapeutic EH inhibition.

Original languageEnglish (US)
Pages (from-to)589-599
Number of pages11
JournalCurrent Pharmaceutical Biotechnology
Volume10
Issue number6
DOIs
StatePublished - 2009

Fingerprint

Epoxide Hydrolases
Enzyme Inhibitors
Tryptophan
Catalytic Domain
Fluorescence
Inhibitory Concentration 50
Enzymes
Enzyme Assays
Calibration
Limit of Detection
Cardiovascular Diseases
Technology

Keywords

  • Enzyme inhibitors
  • Inhibitor detection
  • Soluble epoxide hydrolase
  • Tryptophan fluorescence quenching

ASJC Scopus subject areas

  • Pharmaceutical Science
  • Biotechnology

Cite this

Tryptophan fluorescence quenching by enzyme inhibitors as a tool for enzyme active site structure investigation : Epoxide hydrolase. / Matveeva, Evgenia G.; Morisseau, Christophe; Goodrow, Marvin H.; Mullin, Chris; Hammock, Bruce D.

In: Current Pharmaceutical Biotechnology, Vol. 10, No. 6, 2009, p. 589-599.

Research output: Contribution to journalArticle

Matveeva, Evgenia G. ; Morisseau, Christophe ; Goodrow, Marvin H. ; Mullin, Chris ; Hammock, Bruce D. / Tryptophan fluorescence quenching by enzyme inhibitors as a tool for enzyme active site structure investigation : Epoxide hydrolase. In: Current Pharmaceutical Biotechnology. 2009 ; Vol. 10, No. 6. pp. 589-599.
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AB - We present the strong fluorescence effect, a new 392 nm emission peak appearing after binding of a naphtolurea inhibitor XIIa to the enzyme epoxide hydrolase (EH), along with the quenching of the EH tryptophan fluorescence. We have studied the quenching of the 392-nm peak (attributed to XIIa bound inside the active center of the enzyme) of the mixture EH+XIIa by various strong transparent inhibitors (competing with XIIa for binding to EH), and measured the corresponding values of the Stern-Volmer constants, K(mix)SV. Strong EH inhibitors demonstrate different replacement behavior which can be used to distinguish them. We further demonstrate a novel fluorescent assay which allows to distinguish highly potent inhibitors and to visualize the strongest among them. We generated our assay calibration curve based on the quenching data, by plotting quenching strength K(mix)SV versus inhibiting strength, IC50 values. We used moderate inhibitors for the assay plot generation. We then applied this curve to determine IC50 values for several highly potent inhibitors, with IC50 values at the limit of the IC50 detection sensitivity by colorimetric enzyme assay. IC 50 values determined from our quenching assay show correlation with IC50 values determined in the literature by more sensitive radioactive-based assay and allow differentiating the inhibitors potency in this group. To our knowledge, this is the first inhibitor assay of such kind. Chemical inhibition of EH is an important technology in the treatment of various cardiovascular diseases, therefore, this tool may play a crucial role in discovering new inhibitor structures for therapeutic EH inhibition.

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