SITE-DIRECTED MUTAGENESIS OF HUMAN GLUTATHIONE TRANSFERASE P1-1 - SPECTRAL, KINETIC, AND STRUCTURAL-PROPERTIES OF CYS-47 AND LYS-54 MUTANTS

In the human placental glutathione transferase, Cys 47 possesses, at p hysiological pH values, a pK(a) value of 4.2 and may exist as an ion p air with the protonated epsilon-amino group of Lys-54, Using site dire cted mutagenesis we investigate spectral, kinetic, and structural prop erties of Cys-47 and Lys-54 mutants. The results shown indicate that t he thiolate ion detected at 229 nm should be assigned exclusively to C ys-47. The contribution of Lys-54 to the activation of Cys-47 is asses sed by the spectral properties of the K54A mutant enzyme. The induced cooperativity toward glutathione, as a consequence of mutation of Lys- 54 to alanine, clearly parallels that observed for the Cys-47 mutant e nzymes (see the preceding paper (Ricci, G., Lo Bello, M., Caccuri, A. M., Pastore, A., Nuccetelli, M., Parker, M. W,, and Federici, G, (1995 ) J. Biol. Chem. 270, 1243-1248) and points out the importance of this electrostatic interaction in shaping the correct spatial arrangement for the binding of glutathione and in anchoring the flexible helix alp ha 2. When this ion pair is disrupted, by mutation of either residue, the flexibility of this region could be greatly increased, causing hel ix alpha 2 to come in contact with the other subunit and generating a structural communication, which is the basis of the observed cooperati vity.