The reaction of 5,5' dithiobis(2 nitrobenzoic acid) (DTNB) with rabbit muscle glyceraldehyde 3 phosphate dehydrogenase [d glyceraldehyde 3 phosphate: NAD+ oxidoreductase (phosphorylating), EC 220.127.116.11] consists of an initial burst of reaction over several minutes, corresponding to 2 thiols/subunit, followed by a further slow reaction of the 2 remaining groups. The initial fast release of 2 thionitrobenzoate anions per subunit is shown to be due to reaction of two sulphydryl groups with DTNB. One of these reacts very fast, with consequent inactivation of the enzyme, and is therefore assumed to be the active site thiol, Cys 149. A mechanism involving reaction of DTNB with Cys 149, followed by formation of an intramolecular disulphide bridge between Cys 149 and a second thiol, is shown to be unlikely. Incubation of the enzyme with ethacrynic acid abolishes the initial fast reaction with DTNB of 2 groups/subunit, and 2 ethacrynic residues/subunit remain attached to the enzyme after thorough dialysis. Ethacrynic acid treatment results in inactivation of the enzyme, which is not reversed by dilution or by addition of 2 mercaptoethanol. Furthermore, the reactivity of the 2 sulphydryl groups blocked by ethacrynic acid is not restored by denaturation or by exhaustive dialysis. One thiol per subunit reacts rapidly with ethacrynic acid, and one more slowly. The rate of inactivation of the enzyme correlates with the rate of reaction of the fast group, which is assumed to be the active site thiol. Increasing the extent of saturation of the enzyme with NAD+ results in protection against inactivation by ethacrynic acid. The data suggest that ethacrynic acid reacts with 2 thiols/enzyme subunit and that inactivation of the enzyme by the drug results from modification of the active site thiol.
|Number of pages||10|
|Publication status||Published - 1 Mar 1973|