Inhibition of Escherichia coli β-galactosidase by 2-nitro-1-(4,5-dimethoxy-2-nitrophenyl) ethyl, a photoreversible thiol label

Rachel Golan, Uri Zehavi*, Michael Naim, Abraham Patchornik, Patricia Smirnoff

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

23 Scopus citations

Abstract

1-Nitro-2-phenylethene (β-nitrostyrene, 1), which is a thiol-protecting reagent (Jung, G., Fouad, H. and Heusel, G. (1975) Angew. Chem. Int. Ed. Engl. 14, 817-818), was demonstrated in this work to be an irreversible inhibitor of β-galactosidase (EC 3.2.1.23), an enzyme known to be inhibited by some thiol reagents or through modifying a methionine residue at the active site. No reversal of the inhibition was observed upon subsequent incubation with mercaptoethanol or irradiation (350 nm). 1-(4,5-dimethoxy-2-nitrophenyl)-2-Nitroethene (2) was also shown to be an irreversible inhibitor (94% inhibition, pH 8.3) of the enzyme. Kcat values of β-galactosidase at pH 8.3 with o-nitrophenyl β-D-galactopyranoside (ONPG) as the substrate and at the highest inhibitor concentrations employed for compound 1 (4.06 · 10-4 M) ranged from 1.67 · 104 s-1 after 30 min of preincubation to < 0,07 · 104 s-1 after 180 min preincubation. For compound 2 (9.5 · 10-5 M) Kcat values ranged from 2.70 · 104 s-1 following 30 min preincubation to 1.15 · 104 s-1 after 180 min of preincubation; the changes in Kappm, however, were small. The activity was not recovered following incubation with mercaptoethanol. Since compound 2 and the inhibited enzyme are 2-nitrobenzyl derivatives, they are expected to be photosensitive and indeed, irradiation of the inhibited enzyme in the presence of mercaptoethanol resulted in recovery (89%, pH 8.3) of the enzyme activity.

Original languageEnglish
Pages (from-to)238-242
Number of pages5
JournalBiochimica et Biophysica Acta - Protein Structure and Molecular Enzymology
Volume1293
Issue number2
DOIs
StatePublished - 16 Apr 1996

Keywords

  • (E. coli)
  • Photoreversible thiol label
  • β-Galactosidase

Fingerprint

Dive into the research topics of 'Inhibition of Escherichia coli β-galactosidase by 2-nitro-1-(4,5-dimethoxy-2-nitrophenyl) ethyl, a photoreversible thiol label'. Together they form a unique fingerprint.

Cite this