Enzymatic defects underlying hereditary glutamate cysteine ligase deficiency are mitigated by association of the catalytic and regulatory subunits.

Glutamate cysteine ligase (GCL) deficiency is a rare autosomal recessive trait that compromises production of glutathione, a critical redox buffer and enzymatic cofactor. Patients have markedly reduced levels of erythrocyte glutathione, leading to hemolytic anemia and, in some cases, impaired neurological function. Human glutamate cysteine ligase is a heterodimer comprised of a catalytic subunit (GCLC) and a regulatory subunit (GCLM), which catalyzes the initial rate-limiting step in glutathione production. Four clinical missense mutations have been identified within GCLC: Arg127Cys, Pro158Leu, His370Leu, and Pro414Leu. Here, we have evaluated the impacts of these mutations on enzymatic function in vivo and in vitro to gain further insight into the pathology. Embryonic fibroblasts from GCLC null mice were transiently transfected with wild-type or mutant GCLC, and cellular glutathione levels were determined. The four mutant transfectants each had significantly lower levels of glutathione relative to that of the wild type, with the Pro414Leu mutant being most compromised. The contributions of the regulatory subunit to GCL activity were investigated using a Saccharomyces cerevisiae model system. Mutant GCLC alone could not complement a glutathione deficient strain and required the concurrent addition of GCLM to restore growth. Kinetic characterizations of the recombinant GCLC mutants indicated that the Arg127Cys, His370Leu, and Pro414Leu mutants have compromised enzymatic activity that can largely be rescued by the addition of GCLM. Interestingly, the Pro158Leu mutant has kinetic constants comparable to those of wild-type GCLC, suggesting that heterodimer formation is needed for stability in vivo. Strategies that promote heterodimer formation and persistence would be effective therapeutics for the treatment of GCL deficiency.