Generation of a Ni(II) binding site by introduction of a histidine cluster in the structure of human glutathione transferase A1-1.

Abstract

Two mutant forms of human glutathione transferase (GST) A1-1 with affinity for metal ions were constructed by introduction of His residues by site-directed mutagenesis. A mutant, 2-His, contained the mutations Lys84Gln, Asp85His and Glu88His, and another, 5-His, contained the mutations Tyr79His, Asn80His, Lys84His, Asp85His and Glu88His. The mutant proteins were obtained in good yields (40-150 mg per 3 l culture) by heterologous expression in Escherichia coli. The mutant enzymes possessed novel binding affinities for Ni(II) and Zn(II) ions, as demonstrated by immobilized metal ion affinity chromatography. The mutant with two novel His residues (2-His mutant) did not bind as tightly to immobilized Ni(II) as did the mutant with five novel His residues (5-His mutant). When tested for affinity to immobilized Zn(II), only the 5-His mutant remained bound to the column. The affinity of the 5-His mutant for Ni(II) ions in solution was determined by binding experiments in an aqueous polymeric two-phase system. Analysis of the binding curve showed two binding sites per enzyme subunit and a dissociation constant of 6.7 +/- 1.6 mu M. The kinetic constants kcat, Km and kcat/Km for the reaction with glutathione and 1-chloro-2,4-dinitrobenzene were determined by steady-state kinetic analysis and the parameter values for the mutant forms were found to be indistinguishable from those obtained for the wild-type GST A1-1. The differences in surface charge in the mutant proteins as compared with the wild-type enzyme did not alter the pH dependence of kcat. The results provide an alternative method for purification of fully active recombinant GST A1-1 by the introduction of novel metal binding sites. The data also showed that two His residues are sufficient for Ni(II) binding.