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Photoaffinity Labeling of Rat Liver Glutathione S-Transferase, 4-4, by Glutathionyl S-[4-(Succinimidyl)-benzophenone]†

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Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware 19716
Cite this: Biochemistry 1998, 37, 45, 15671–15679
Publication Date (Web):October 16, 1998
https://doi.org/10.1021/bi981381k
Copyright © 1998 American Chemical Society
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    Abstract

    Glutathionyl S-[4-(succinimidyl)benzophenone] (GS-Succ-BP), an analogue of the product of glutathione and xenobiotic substrate, was synthesized and shown to act as a photoaffinity label of rat liver glutathione S-transferase, 4-4. A time-dependent photoinactivation occurs upon irradiation at long wavelength UV light of the complex of enzyme and GS-Succ-BP. The rate of inactivation exhibits nonlinear dependence on [GS-Succ-BP], characterized by an apparent KI of 115 μM and kmax of 0.469 min-1. Effective protection against photoinactivation by 150 μM GS-Succ-BP is provided by dinitrophenol, nitrobenzene, ethacrynic acid, and S-hexylglutathione, analogues of xenobiotic substrates and product. These results suggest that GS-Succ-BP reacts with the enzyme within the active site, probably in the xenobiotic substrate-binding site. Upon complete inactivation, reagent incorporation of about 1 mol/mol of enzyme dimer is measured by radioactivity and MALDI-TOF mass spectrometry. Isolation of modified peptides followed by gas-phase sequencing and mass spectrometry indicates that Met-112 is the only reaction target of GS-Succ-BP. Although only one subunit of the enzyme dimer is modified, catalytic activity of both subunits is lost. Molecular modeling suggests that the benzophenone moiety of the compound binds in the cleft between the two enzyme subunits and modification of Met-112 on one subunit excludes reaction of the corresponding methionine on the other subunit. It is proposed that the new compound, glutathionyl S-[4-(succinimidyl)benzophenone], may have general applicability as a photoaffinity label of other enzymes with glutathione binding sites.

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    †

     This work was supported by United States Public Health Service Grant R01 CA66561.

    *

     To whom correspondence should be addressed. Phone:  (302) 831-2973. Fax:  (302) 831-6335. E-mail:  [email protected].

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    17. Y. Han, A. Bisello, C. Nakamoto, M. Rosenblatt, M. Chorev. 3‐(3′‐Fluorenyl‐9′‐oxo)‐ l ‐alanine: a novel photoreactive conformationally constrained amino acid. The Journal of Peptide Research 2000, 55 (3) , 230-239. https://doi.org/10.1034/j.1399-3011.2000.00175.x
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