Role of a topologically conserved Isoleucine in the structure and function of Glutathione Transferases

[摘要] Proteins in the glutathione transferase family share a common fold. The close packing ofsecondary structures in the thioredoxin fold in domain 1 forms a compact hydrophobiccore. This fold has a bababba topology and most proteins/domains with this fold have atopologically conserved isoleucine residue at the N-terminus of a-helix 3. Class Alphaglutathione transferases are one of 12 classes within the glutathione transferase family.To investigate the role of the conserved isoleucine residue in the structure, function andstability of glutathione transferases, homodimeric human glutathione transferase A1-1(hGST A1-1) was used as a representative of the GST family. Ile71 was replaced withvaline and the properties of I71V hGST A1-1 were compared with those of wildtypehGST A1-1. The spectral properties monitored using far-UV CD and tryptophanfluorescence indicated little change in secondary or tertiary structure confirming theabsence of any gross structural changes in hGST A1-1 due to the incorporation of themutation. Both wildtype and mutant dimeric proteins were determined to have amonomeric molecular mass of 26 kDa. The specific activity of I71V hGST A1-1 (130mmol/min/mg) was three times that of wildtype hGST A1-1 (48 mmol/min/mg). I71VhGST A1-1 showed increased kinetic parameters compared to wildtype with a 10-foldincrease in kcat/Km for CDNB. The increase in Km of I71V hGST A1-1 suggests themutation had a negative effect on substrate binding. The DDG for transition statestabilisation was –5.82 kJ/mol which suggest the I71V mutation helps stabilise thetransition state of the SNAR reaction involving the conjugation of reduced glutathione(GSH) to 1-chloro-2,4-dinitrobenzene (CDNB). A 2-fold increase in the IC50 value forI71V hGST A1-1 (11.3 mM) compared to wildtype (5.4 mM) suggests that the mostnoticeable change due to the mutation occurs at the H-site of the active site.Conformational stability studies were performed to determine the contribution of Ile71 toprotein stability. The non-superimposability of I71V hGST A1-1 unfolding curves andthe decreased m-value suggest the formation of an intermediate state. The conformationalstability of I71V hGST A1-1 (16.5 kcal/mol) was reduced when compared to that of thewildtype (23 kcal/mol). ITC was used to dissect the binding energetics of Shexylglutathioneto wildtype and I71V hGSTA1-1. The ligand binds 5-fold more tightlyto wildtype hGST A1-1 (0.07 mM) than I71V hGST A1-1 (0.37 mM). The I71V mutant displays a larger negative DCp than wildtype hGST A1-1 (DDCp = -0.41 kJ/mol/K). Thisindicates that a larger solvent-exposed hydrophobic surface area is buried for I71V hGSTA1-1 than for wildtype hGST A1-1 upon the binding of S-hexylglutathione. Overall theresults suggest that Ile71 conservation is for the stability of the protein as well as playinga pivotal indirect role in catalysis and substrate binding.

[发布机构] University of the Witwatersrand