Naturally occurring human glutathione S-transferase GSTP1-1 isoforms with isoleucine and valine in position 104 differ in enzymic properties.
Glutathione S-transferase P1-1 isoforms, differing in a single amino acid residue (Ile104 or Val104), have been previously identified in human placenta [Ahmad, H., Wilson, D. E., Fritz, R. R., Singh, S. V., Medh, R. D., Nagle, G. T., Awasthi, Y. C. & Kurosky, A. (1990) Arch. Biochem. Biophys. 278, 398-408]. In the present report, the enzymic properties of these two proteins are compared. [I104]glutathione S-transferase P1-1 has been expressed from its cDNA in Escherichia coli and purified to homogeneity by affinity chromatography; the cDNA has been mutated 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 (KM values for 1-chloro-2,4-dinitrobenzene were 0.8 mM and 3.0 mM for [I-104]glutathione S-transferase P1-1 and [V-104]glutathione S-transferase P1-1, respectively), but were identical in their affinity for glutathione. In addition, the two enzymes were distinguishable by their heat stability, with half-lives 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, indicate that the residue in position 104 helps to define the geometry of the hydrophobic substrate-binding site, and may also influence activity by interacting with residues directly involved in substrate binding.
Zimniak
P
P
Department of Medicine, University of Arkansas for Medical Sciences, Little Rock.
Nanduri
B
B
Pikuła
S
S
Bandorowicz-Pikuła
J
J
Singhal
S S
SS
Srivastava
S K
SK
Awasthi
S
S
Awasthi
Y C
YC
D001483:Base Sequence; D002846:Chromatography, Affinity; D004795:Enzyme Stability; D005982:Glutathione Transferase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006358:Hot Temperature; D006801:Humans; D007532:Isoleucine / Q000378:metabolism*; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D013379:Substrate Specificity; D014633:Valine / Q000378:metabolism*
D009838:Oligodeoxyribonucleotides; D007532:Isoleucine; D005982:Glutathione Transferase; D014633:Valine