Mechanism of aging of rat muscle glyceraldehyde-3-phosphate dehydrogenase studied by selective enzyme-oxidation

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Controlled oxidation of rat muscle glyceraldehyde-3-phosphate dehydrogenase (GPDH) was carried out in an attempt to simulate age-related effects observed in enzyme samples purified from old animals. A comparative study of the “simulated aged” and of native young and old GPDH forms was done using fluorescence techniques. The present work is based on our previous findings that the locus of the age-related modifications in GPDH is in the nicotinamide-binding site, where the catalytically active Cys-149 residue is located, and that an increase in oxidation potential occurs in old animal tissues which may enable various oxidizing agents to play a significant role in the inactivation of certain enzymes. Thus it has been suggested that the loss of specific activity observed in old GPDH may be due to subtle and irreversible conformational changes caused by reaction of Cys-149 with these agents. The circularly polarized luminescence (CPL) spectrum emitted by the fluorescent sulfhydryl reagent I-AEDANS covalently bound to GPDH through Cys-149 at the nicotinamide binding site, revealed a significant difference in conformation between these sites in young and old GPDH forms. Large differences were also observed between corresponding spectra when the binding sites were saturated with NAD +, reflecting the development of marked conformational changes in both young and old GPDH species upon coenzyme binding. The oxidizing reagents employed in the current study (hydrogen peroxide, superoxide radical and atmospheric dioxygen) are all expected to be more commonly encountered in the less reducing environment of old animal tissues. All of them, though to a different extent, caused a significant inactivation of the enzyme dependent on the initial oxidant concentration. Although the original enzymatic activity could be partially restored by incubation with a reducing agent, the prior oxidation was found to induce some irreversible structural changes as expressed in a decrease in the number of fast reacting SH groups. The extent of irreversible inactivation was a function of both oxidant concentration and the duration of exposure to the oxidant. The affinity of the oxidized GPDH species (termed “aged”) toward coenzyme, as monitored by fluorometric titrations, was markedly lower than that observed for both the native young and old GPDHs. In addition, the CPL spectra of the “aged” enzymes were different from those obtained for both native forms. This indicates that the structural modifications induced by the oxidation reactions tested differ from those present in native old GPDH, although in each case the changes are localized within the nicotinamide binding site.