Studies on the mechanism of oxidative modification of human glyceraldehyde-3-phosphate dehydrogenase by glutathione: catalysis by glutaredoxin.

In this report the protein human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has been examined to clarify the roles of (a) direct oxidation and (b) thiol-disulphide exchange (with glutathione disulphide) on the modification of its catalytic activity. An in vitro system using purified human GAPDH and [35S]-GSSG (glutathione disulphide), has permitted clarification of these possibilities by showing that S-glutathionylation of GAPDH does not result in an inactivation of the enzyme. Rather, the direct oxidation of GAPDH with hydrogen peroxide is responsible for inhibition of the catalytic activity of the protein. Furthermore, pre-treatment of the enzyme with hydrogen peroxide enhances the formation of glutathione-GAPDH mixed disulphides in the presence of glutathione disulphide. This may serve as a molecular "switch" directing the protein to other reported functions in the cell. It is also shown that the efficiency of S-glutathionylation of either native or oxidised GAPDH is enhanced by the presence of recombinant glutaredoxin (thiol transferase) of either bacterial or human origin. Under the conditions of analysis the glutaredoxin itself is also shown to readily undergo S-glutathionylation external to its active site. Taken together, the data indicate the complexity of mechanisms likely to be involved in regulating cellular proteins during oxidative stress and implicate controlled enzyme-catalysed S-glutathionylation as a potential selectivity factor in the redox modification of protein function by glutathione.