Theresa Mitchell

N,N'-o-Phenylenedi[1,4-14C]maleimide. Its synthesis and application in the cross-linking of Escherichia coli succinic thiokinase.

Abstract 1. 1. The syntheses of N , N ′- o -phenylenedi[1,4- 14 C]maleimide (60–70% yield) and the unlabeled ortho and para isomers (40–50% yield) have been carried out on a milligram scale. 2. 2. Use of the 14 C-labeled compound with Escherichia coli succinic thiokinase showed that the crosslinking agent combined with the β subunit first in the formation of αβ dimer. Incorporation of about 2.5 moles of the reagent (40% of this bound bifunctionally) was sufficient to inhibit enzyme activity completely.

Inactivation of Escherichia coli succinic thiokinase by selective oxidation of thiol groups by permanganate

Succinic thiokinase from Escherichia coli was rapidly inactivated by permanganate ion at 25° and 0°. On the basis of the cysteic acid content of hydrolysates of treated protein, oxidation of 3 sulfhydryl groups appeared to effect total loss of thiokinase activity. However, titration of the same protein samples revealed that 4 important sulfhydryl groups (a fraction of which was possibly in disulfide form) were more likely oxidized during the inactivation process. Significant protection of the enzyme against permanganate inactivation was obtained by the following additions: ATP-Mg2+ and succinate (51%); desulfo-CoA alone (53%); and ATP-Mg2+, succinate, and desulfo-CoA (93%). No protection was observed when either inorganic phosphate or arsenate was added.