GENERATION OF A NI(II) BINDING-SITE BY INTRODUCTION OF A HISTIDINE CLUSTER IN THE STRUCTURE OF HUMAN GLUTATHIONE TRANSFERASE A1-1

Two mutant forms of human glutathione transferase (GST) A1-1 with affi nity for metal ions were constructed by introduction of His residues b y site-directed mutagenesis. A mutant, 2-His, contained the mutations Lys84Gln, Asp85His and Glu88His, and another, 5-His, contained the mut ations Tyr79His, Asn80His, Lys84His, Asp85His and Glu88His, The mutant proteins were obtained in good yields (40-150 mg per 3 1 culture) by heterologous expression in Escherichia coli, The mutant enzymes posses sed novel binding affinities for Ni(II) and Zn(II) ions, as demonstrat ed by immobilized metal ion affinity chromatography, The mutant with t wo novel His residues (2-His mutant) did not bind as tightly to immobi lized Ni(II) as did the mutant with five novel His residues (5-His mut ant), When tested for affinity to immobilized Zn(II), only the 5-His m utant remained bound to the column, The affinity of the 5-His mutant f or Ni(II) ions in solution was determined by binding experiments in an aqueous polymeric two-phase system, Analysis of the binding curve sho wed two binding sites per enzyme subunit and a dissociation constant o f 6.7 +/- 1.6 mu M The kinetic constants k(cat), K-m and k(cat)/K-m fo r the reaction with glutathione and 1-chloro-2,4-dinitrobenzene were d etermined by steady-state kinetic analysis and the parameter values fo r the mutant forms were found to be indistinguishable from those obtai ned for the wild-type GST A1-1, The differences in surface charge in t he mutant proteins as compared with the wild-type enzyme did not alter the pH dependence of k(cat). The results provide an alternative metho d for purification of fully active recombinant GST A1-1 by the introdu ction of novel metal binding sites, The data also showed that two His residues are sufficient for Ni(II) binding.