The reactions of Escherichia coli citrate synthase with the sulfhydryl reagents 5,5′-dithiobis-(2-nitrobenzoic acid) and 4,4′-dithiodipyridine

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Abstract
Citrate synthase of E. coli reacts rapidly with 1 equivalent of Ellman''s reagent, 5,5''-dithiobis-(2-nitrobenzoic acid)(DTNB), per subunit, losing completely its sensitivity to the allosteric inhibitor, NADH. When the enzyme is treated instead with 4,4''-dithiodipyridine (4,4''-PDS), all activity is lost. Certain evidence shows that the SH group modified by DTNB, and that whose modification by 4,4''-PDS inactivates the enzyme, are the same. Both reagents abolish NADH fluorescence enhancement by the enzyme. Saturating levels of NADH and some other adenyl acid derivatives inhibit the reactions with both reagents. When the enzyme is modified with 1 equivalent of DTNB or 4,4''-PDS, subsequent reactivity toward the other reagent is greatly decreased. Following modification, the DTNB and 4,4''-PDS derivatives spontaneously lose thionitrobenzoate (TNB) or pyridine-4-thione (PT), respectively, in reactions which are thought to involve displacement of TNB or PT by a 2nd enzyme SH group, so that an enzyme disulfide is introduced. The introduction of the disulfide bond, if this is what occurs, does not lead to cross-linking of citrate synthase polypeptide chains, as judged by sodium dodecyl sulfate polyacrylamide gel electrophoresis under nonreducing conditions. Certain evidence was also found that the sites of modification by DTNB and 4,4''-PDS are not the same. DTNB modification desensitizes to NADH but does not inactivate, while 4,4''-PDS inactivates at least 99.9%. The presumed disulfide from elimination of TNB is also active, while that from PT modification is no more active than the original 4,4''-PDS modified product. Prior modification of the enzyme with DTNB affords no protection against later inactivation by 4,4''-PDS. The studies therefore indicate a close relationship between the DTNB desensitization and 4,4''-PDS inactivation, but they are unable to identify it exactly. Other properties of the DTNB reaction are also described, and a hypothesis is offered to explain quantitatively the finding that desensitization lags behind modification during the modification of citrate synthase by DTNB.

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