ENGINEERING OF A METAL COORDINATING SITE INTO HUMAN GLUTATHIONE TRANSFERASE M1-1 BASED ON IMMOBILIZED METAL-ION AFFINITY-CHROMATOGRAPHY OF HOMOLOGOUS RAT ENZYMES

Rat glutathione transferase (GST) 3-3 binds to Ni(II)iminodiacetic aci d (IDA)-agarose, whereas other GSTs that are abundant in rat liver do not bind to this immobilized metal ion affinity chromatography (IMAC) adsorbent. Rat GST 3-3 contains two superficially located amino acid r esidues, His84 and His85, that are suitably positioned for coordinatio n to Ni(II)-IDA-agarose. This particular structural motif is lacking i n GSTs that do not bind to the IMAC matrix. Creation of an equivalent His-His structure in the homologous human GST M1-1 by protein engineer ing afforded a mutant enzyme that displays affinity for Ni(II)IDA-agar ose, in contrast to the wild-type GST M1-1. The results identify a dis tinct site that is operational in IMAC and suggest an approach to the rational design of novel integral metal coordination sites in proteins .