Combinatorial chemical reengineering of the alpha class glutathione transferases.
Previously, we discovered that human glutathione transferases (hGSTs) from the alpha class can be rapidly and quantitatively modified on a single tyrosine residue (Y9) using thioesters of glutathione (GS-thioesters) as acylating reagents. The current work was aimed at exploring the potential of this site-directed acylation using a combinatorial approach, and for this purpose a panel of 17 GS-thioesters were synthesized in parallel and used in screening experiments with the isoforms hGSTs A1-1, A2-2, A3-3, and A4-4. Through analytical HPLC and MALDI-MS experiments, we found that between 70 and 80% of the reagents are accepted and this is thus a very versatile reaction. The range of ligands that can be used to covalently reprogram these proteins is now expanded to include functionalities such as fluorescent groups, a photochemical probe, and an aldehyde as a handle for further chemical derivatization. This site-specific modification reaction thus allows us to create novel functional proteins with a great variety of artificial chemical groups in order to, for example, specifically tag GSTs in biological samples or create novel enzymatic function using appropriate GS-thioesters.
Viljanen
Johan
J
IFM, Department of Organic Chemistry, Linköping University, S-581 83, Sweden.
Tegler
Lotta
L
Broo
Kerstin S
KS
D000215:Acylation; D019817:Benzoic Acid / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D020650:Combinatorial Chemistry Techniques / Q000379:methods*; D018360:Crystallography, X-Ray; D004952:Esters / Q000737:chemistry; D005982:Glutathione Transferase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D008958:Models, Molecular; D015394:Molecular Structure; D015202:Protein Engineering / Q000379:methods*; D017434:Protein Structure, Tertiary; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013438:Sulfhydryl Compounds / Q000737:chemistry
D004952:Esters; D013438:Sulfhydryl Compounds; D019817:Benzoic Acid; D005982:Glutathione Transferase