Protection of phosphoglycerate kinase against in vitro aging by selective cysteine methylation.
Recent studies have demonstrated that the aging effects in phosphoglycerate kinase (PGK) may be simulated in vitro by prolonged incubation of the enzyme under nonreducing conditions followed by reduction with excess 2-mercaptoethanol. The simulated-old enzyme thus produced appears to be identical to native old PGK and, like the latter enzyme, may be successfully rejuvenated by an unfolding-refolding procedure. A model for PGK aging was proposed in which initial and reversible oxidation of the enzyme is followed by conformational modifications that persist after the enzyme is re-reduced. The role of specific cysteine oxidation in the initial step of PGK aging was tested in the present study by selectively methylating the fast-reacting cysteine residues in this enzyme, thus blocking the putative oxidation sites, and producing in vitro a young form of PGK that is immune to aging. The methylation was performed by treating the enzyme with excess iodomethane and monitoring the reaction by determining the concentration of unreacted cysteines in the enzyme as a function of time. Unmethylated controls were incubated similarly but in the absence of iodomethane. The methylated as well as control samples of PGK were subsequently incubated under conditions which caused native young PGK to develop the age-related effects and become identical to native old PGK. In contrast, the methylated enzyme remained identical to young PGK. These findings strongly support the hypothesis that cysteine oxidation is an essential step in the aging of rat muscle phosphoglycerate kinase.
Cook
L L
LL
Institute of Gerontology, University of Michigan, Ann Arbor 48109.
Gafni
A
A
D000375:Aging; D000818:Animals; D003545:Cysteine; D007700:Kinetics; D008623:Mercaptoethanol / Q000494:pharmacology*; D008745:Methylation; D024510:Muscle Development; D009132:Muscles / Q000201:enzymology; D010735:Phosphoglycerate Kinase / Q000378:metabolism*; D051381:Rats; D011916:Rats, Inbred F344; D012016:Reference Values
D008623:Mercaptoethanol; D010735:Phosphoglycerate Kinase; D003545:Cysteine