The goal of this research has been to identify factors which influence changes in the properties of cellular components as a function of age. Free radicals generated in vivo have the potential to damage cellular components. This process has been implicated in the age-related impairment of cellular function. In vitro studies designed to assess the susceptibility of key metabolic enzymes to oxidative conditions known to exist in vivo are therefore critical to the understanding of aging. Metal-catalyzed production of free radicals has been shown to result in site-specific modification and inactivation of various enzymes. We have previously reported that glucose-6-phosphate dehydrogenase (Glu-6-PDH) from Leuconostoc mesenteroides is rapidly inactivated by ferrous iron and hydrogen peroxide in a site-specific manner. In an extension of this work, we have found that incubation of this enzyme with ferrous iron and citrate results in oxygen-dependent inactivation of the enzyme. Our data suggest that the ferrous iron-citrate complex binds to the glucose-6- phosphate binding site and then undergoes reaction with hydrogen peroxide formed in solution leading to the oxidative modification of amino acids essential for enzyme activity. Reactive oxygen species are also known to readily interact with membrane lipids, often resulting in the formation of unsaturated aldehydes. These aldehydes are likely to diffuse into cellular media where they are available for facile reaction with a vast array of bimolecules. 4- Hydroxy-2-nonenal (HNE) is one of the major products of membrane peroxidation. We have found that incubation of Glu-6-PDH with HNE leads to a pseudo-first-order loss of enzyme activity. Our data indicate that inactivation of Glu-6-PDH by HNE is the result of the reaction of HNE with the epsilon-amino group of a lysine residue involved in the binding of glucose-6-phosphate. Our results demonstrate the inherent susceptibility of Glu-6-PDH from L. mesenteroides to oxidative conditions known to exist in vivo and identify likely products resulting from oxidative stress. These studies therefore provide valuable information for current research which focuses on in vivo experiments designed to directly assess the role of these processes in the age-related impairment of cellular function.
