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 physiological pH values, a pK alpha value of 4.2 and may exist as an ion pair with the protonated epsilon-amino group of Lys-54. Using site-directed mutagenesis we investigate spectral, kinetic, and structural properties of Cys-47 and Lys-54 mutants. The results shown indicate that the thiolate ion detected at 229 nm should be assigned exclusively to Cys-47. The contribution of Lys-54 to the activation of Cys-47 is assessed 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 enzymes (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 alpha 2. When this ion pair is disrupted, by mutation of either residue, the flexibility of this region could be greatly increased, causing helix alpha 2 to come in contact with the other subunit and generating a structural communication, which is the basis of the observed cooperativity.
Department of Biology, University of Rome Tor Vergata, Rome, Italy.
M
M
Lo Bello
A
A
Battistoni
A P
AP
Mazzetti
P G
PG
Board
M
M
Muramatsu
G
G
Federici
G
G
Ricci
D001483:Base Sequence; D003545:Cysteine / Q000737:chemistry*; D004795:Enzyme Stability; D005982:Glutathione Transferase / Q000737:chemistry* / Q000235:genetics; D006801:Humans; D006868:Hydrolysis; D007527:Isoenzymes / Q000737:chemistry*; D007700:Kinetics; D008239:Lysine / Q000737:chemistry*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D011487:Protein Conformation; D013056:Spectrophotometry, Ultraviolet
D007527:Isoenzymes; D009838:Oligodeoxyribonucleotides; D005982:Glutathione Transferase; D008239:Lysine; D003545:Cysteine