کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
1930408 | 1050510 | 2011 | 7 صفحه PDF | دانلود رایگان |
![عکس صفحه اول مقاله: Aspartate 458 of human glutathione synthetase is important for cooperativity and active site structure Aspartate 458 of human glutathione synthetase is important for cooperativity and active site structure](/preview/png/1930408.png)
Human glutathione synthetase (hGS) catalyzes the second ATP-dependent step in the biosynthesis of glutathione (GSH) and is negatively cooperative to the γ-glutamyl substrate. The hGS active site is composed of three highly conserved catalytic loops, notably the alanine rich A-loop. Experimental and computational investigations of the impact of mutation of Asp458 are reported, and thus the role of this A-loop residue on hGS structure, activity, negativity cooperativity and stability is defined. Several Asp458 hGS mutants (D458A, D458N and D458R) were constructed using site-directed mutagenesis and their activities determined (10%, 15% and 7% of wild-type hGS, respectively). The Michaelis–Menten constant (Km) was determined for all three substrates (glycine, GAB and ATP): glycine Km increased by 30–115-fold, GAB Km decreased by 8–17-fold, and the ATP Km was unchanged. All Asp458 mutants display a change in cooperativity from negative cooperativity to non-cooperative. All mutants show similar stability as compared to wild-type hGS, as determined by differential scanning calorimetry. The findings indicate that Asp458 is essential for hGS catalysis and that it impacts the allostery of hGS.
► Mutations of Asp458 to alanine, asparagine and arginine have a large impact on human glutathione synthetase (hGS) activity.
► The affinities of GAB and glycine substrates are different in Asp458 mutant hGS.
► Mutations at Asp458 impact the allosteric pathway of hGS causing a loss of cooperativity.
► Asp458Asn (D458N) exhibits an increase in activity and glycine affinity after prolonged storage.
► Asp458 mutant enzymes show no loss in thermal stability and secondary structure is unaffected.
Journal: Biochemical and Biophysical Research Communications - Volume 411, Issue 3, 5 August 2011, Pages 536–542