NATURALLY-OCCURRING HUMAN GLUTATHIONE-S-TRANSFERASE GSTP1-1 ISOFORMS WITH ISOLEUCINE AND VALINE IN POSITION-104 DIFFER IN ENZYMATIC-PROPERTIES

Glutathione S-transferase p1-1 isoforms, differing in a single amino a cid residue (Ile104 or Val104), have been previously identified in hum an placenta [Ahmad, H., Wilson, D. E., Fritz, R. R., Singh, S. V., Med h, R. D., Nagle, G. T., Awasthi, Y. C. and Kurosky, A. (1990) Arch, Bi ochem. Biophys. 278, 398-408]. In the present report, the enzymic prop erties of these two proteins are compared. [Il04]glutathione S-transfe rase P1-1 has been expressed from its cDNA in Escherichia coli and pur ified to homogeneity by affinity, chromatography; the cDNA has been mu tated to replace Ile104 by Val104, and [V104]glutathione S-transferase P1-1 was expressed and isolated as described for [I104]glutathione S- transferase P1-1. The two enzymes differed in their specific activity and affinity for electrophilic substrates (K-M values for 1-chloro-2,4 -dinitrobenzene were 0.8 mM and 3.0 mM for [I-104]glutathione S-transf erase p1-1 and [V-104]glutathione S-transferase P1-1, respectively), b ut were identical in their affinity for glutathione. In addition, the two enzymes were distinguishable by their heat stability, with half-li ves at 45 degrees C of 19 min and 51 min, respectively. The resistance to heat denaturation was differentially modulated by the presence of substrates. These data, in conjunction with molecular modeling, indica te that the residue in position 104 helps to define the geometry of th e hydrophobic substrate-binding site, and may also influence activity by interacting with residues directly involved in substrate binding.