The high levels of both enzymes of glutathione synthesis found in the infant human lens rapidly reached lower levels by age 10, and thereafter the rate of decrease diminished. Glutathione synthetase activity in the 6 month old lens was six-fold (units/g lens), four-fold (units/mg soluble protein) and two-fold (units/lens) higher than that in the 83 year old, clear human lens. gamma-Glutamylcysteine synthetase activity in the 6 month old lens was sixteen-fold (units/g lens), ten-fold (units/mg soluble protein) and six-fold (units/lens) higher than that in the 83 year old, clear human lens. When lenses from the young adult beagle, rabbit, bovine, and humans are compared, glutathione synthetase activity (units/g lens) varies by about two-fold. gamma-Glutamylcysteine synthetase activity (units/g lens) is quite similar in the first three species, whereas the enzyme activity is more than a magnitude less in young adult human lenses, and becomes much less with increasing age and in a high proportion of life-support system organ donors. The enzyme activity was undetectable in a few of the latter lenses. Loss of activity was not due to increased susceptibility to heat denaturation. The low levels of the enzyme, and total loss in some situations, suggest that gamma-glutamylcysteine synthetase may be an Achilles' Heel of human lens metabolism.
Steady-state kinetic analysis shows that glutathione binds reversibly to both Mg . enzyme and Mg . enzyme. Lglutamate forms of y-glutamylcysteine synthetase to form inactive complexes. The Ki values for binding to these two species of enzyme are 4 mM and 0.4 mM, respectively; those for S-methyl glutathione are 16 mM and 0.5 mM, respectively, These data suggest that glutathione is an important feedback inhibitor and contributes to the regulation of glutathione synthesis by modulating the synthesis rate of the precursor dipeptide. Adenosine 5'-diphosphate (5'ADP) is also an inhibitor and competes with both ATP and L-8-chloroalanine for Mg . enzyme. Lglutamate and Mg . enzyme. L-glutamylphosphate, respectively. Under physiological conditions in the lens, S'ADP competes effectively with 2-cysteine for Mg . enzyme. L-glutamylphosphate, owing to the low concentration of Lcysteine, and less effectively with ATP for Mg. enzyme-L-glutamate, because of a high concentration of ATP.Glutathione, or L-y-glutamylcysteinylglycine, is a major constituent of the ocular lens epithelium and cortex, where it is found in the highest concentrations of any known tissue [I]. The opaque (cataractous) lens has a paucity of glutathione. The enzymic synthesis and the control of that synthesis in vivo are therefore of much interest. The synthesis occurs in two stages in which a y-glutamyl peptide bond, and then an apeptide bond are formed sequentially by y-glutamylcysteine synthetase (EC 6.3.2.2) and glutathione synthetase (EC-6.3.2.3), respectively.The mechanism of the y-glutamylcysteine synthetase-catalyzed reaction in liver involves formation of L-y-glutamylphosphoanhydride as an intermediate [2]. Additionally, some enzyme-ligand interactions and certain half-reactions have been described utilizing enzyme from human erythrocytes [3] and rat kidney [4], respectively. Isotope exchange studies with enzyme from rat kidney and kinetic analysis of enzyme from bovine erythrocytes suggest a random addition of substrates [4a, 51 with the formation of an enzyme-substrate quarternary complex prior to release of products [5]. Studies from this laboratory [6] based upon steady-state kinetic analysis propose that y-glutamylcysteine synthetase from bovine lens catalyzes the reaction by an ordered bi-uni-uni-bi ping-pong sequence in which L-glutamate is the first substrate to bind to the enzyme as shown in Scheme 1 . Those studies have suggested that the enzyme occurs as a Mg . enzyme complex, rather than as a free enzyme.High concentrations of inorganic phosphate result in negligible inhibition [6]. The rate equation (Eq. 1) is as shown previously [6] except that it has been modified to include the inhibitory terms resulting from the presence of glutathione (GSH) and all the inhibitor terms containing phosphate concentration have been omitted due to the lack of phosphate binding to enzyme species. The various inhibition constants, Ki, and association constants, Ka, are separated out so that the other constants can be limited to five 4 values.Ahhvcviatio...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.