text: Enzymatic defects underlying hereditary glutamate cysteine ligase deficiency are mitigated by association of the catalytic and regulatory subunits. - PubMed - NCBI
  1 selected item: 21657237
  Biochemistry. 2011 Jul 26;50(29):6508-17. doi: 10.1021/bi200708w. Epub 2011 Jun 29.
  Willis MN1, Liu Y, Biterova EI, Simpson MA, Kim H, Lee J, Barycki JJ.
  PMC3142541
  Homology model of human GCLC reveals the locations of the four clinical mutations
  The overall model of hGCLC is shown in ribbon representation with β-strands colored in green, α-helices in blue, and loop regions in grey. ADP and the transition state analogue, phosphorylated BSO (BSO-P), were docked into the previously described model and are shown in ball and stick representation. Carbon atoms are colored in grey, oxygen atoms in red, sulfur atoms in yellow, nitrogen atoms in blue, phosphorus in orange, and magnesium atoms in purple. The locations of the four clinical mutations are highlighted, with carbon atoms colored in purple.
  Biochemistry. ;50(29):6508-6517.
  Glutathione production is reduced as a result of each of the four clinical mutations
  Embryonic fibroblasts obtained from GCLC null mice were transiently transfected with wild-type or mutant human GCLC. Inset. hGCLC expression was monitored via western analysis of whole cell lysates. Non-transfected cells (NT) served as a negative control. Main. Total glutathione levels were measured and normalized to protein levels as determined by western analysis (Inset), using β-tubulin (not shown) as a loading control. Mean ± SD values for three replicates are plotted. An ANOVA one-way test with Dunnett's Multiple Comparison Test was used to analyze the data. Untransfected and Pro414Leu transfected cells had glutathione levels that were statistically reduced relative to wild-type GCLC, and are indicated by an asterisk.
  The modifer subunit enhances the activity of wild-type and mutant hGCLC
  An S. cerevisiae strain deficient in glutamate cysteine ligase (Δgsh1) was transformed with wild-type or mutant GCLC, with or without wild-type GCLM. An empty p416ADH vector was co-transformed with the GCLC alone samples. Transformants were restreaked, grown overnight in uracil/leucine deficient media, diluted to 0.2, 0.1, and 0.04 OD600 nm, spotted on uracil/leucine deficient plates, and grown for 36 hours as described in the text.
  Clinical mutations likely disrupt hydrogen bond networks that stabilize the enzyme active site
  The figure illustrates the hydrogen bond networks centered about three of the clinical point mutations. Atoms are colored as in with potential hydrogen bonds illustrated as solid black lines. The three bound magnesium atoms are labeled M1, M2, and M3. Panel A. Arg 127 stabilizes two β-strands within the active site, forming a salt bridge with Asp 49 and a hydrogen bond with the backbone carbonyl of Gly 104. Panel B. His 370 is involved in a hydrogen bond network that positions a β-strand containing Lys 412. Pro 414 is the second of four proline residues in a rigid loop immediately adjacent to Lys 412, which is involved in orienting the β-phosphate of ATP.
  Clinical mutations decrease GCLC protein stability
  Wild-type and mutant GCLC were incubated at 37 ºC and relative enzymatic activity was monitored as a function of time in the absence (white bars) or presence (black bars) of hGCLM. During the 24 hour time course, wild-type enzyme did not show a significant loss of activity (). Panel A. Arg127Cys. Panel B. Pro158Leu. Panel C. His370Leu. Panel D. Pro414Leu.
  Human GCLC clinical mutants have reduced thermal stability that can be restored by ligand binding or heterodimer formation
  Using a ThermoFluor assay, thermal denaturation profiles of wild-type and mutant GCLC were collected, and then plotted as a first derivative curve to determine Tm values (Panels A, C, E). Data were obtained for the apoenzyme (Panel A), the BSO-inhibited enzyme (Panel C), and the GCLC/GCLM heterodimer (Panel E), with average Tm values plotted in Panels B, D, and F, respectively.