Inhibition of Escherichia coli beta-galactosidase by 2-nitro-1-(4,5-dimethoxy-2-nitrophenyl) ethyl, a photoreversible thiol label
Inhibition of Escherichia coli beta-galactosidase by 2-nitro-1-(4,5-dimethoxy-2-nitrophenyl) ethyl, a photoreversible thiol label
复制标题
DOI：
10.1016/0167-4838(95)00254-5
复制
发表时间：
1996-04-16
期刊：
影响因子：
--
通讯作者：
Smirnoff, P
中科院分区：
文献类型：
--
作者：
Golan, R;Zehavi, U;Smirnoff, P
1-Nitro-2-phenylethene (beta-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 beta-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. K-cat values of beta-galactosidase at pH 8.3 with o-nitrophenyl beta-D-glaacctoyranoside (ONPG) as the substrate and at the highest inhibitor concentrations employed for compound 1 (4.06 . 10(-4) M) ranged from 1.67 . 10(4) s(-1) after 30 min of preincubation to < 0.07 . 10(4) s(-1) after 180 min preincubation, For compound 2 (9.5 . 10(-5) M) K-cat values ranged from 2.70 . 10(4) s(-1) following 30 min preincubation to 1.15 . 10(4) s(-1) after 180 min of preincubation; the changes in K-m(app), 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.
