Inhibition of glutathione transferase pi from human placenta by 1-chloro-2,4-dinitrobenzene occurs because of covalent reaction with cysteine 47.
Human placenta glutathione transferase pi is irreversibly inhibited when incubated with 1-chloro-2,4-dinitrobenzene (CDNB) in the absence of the cosubstrate glutathione. The enzyme is protected against CDNB inactivation by the presence of S-methylglutathione and glutathione. The kinetics of inactivation is pseudo-first-order with k(obs) = 0.04 min-1 when 44 microM enzyme is incubated in presence of 1 mM CDNB at pH 6.5. The inhibition is saturable with respect to the CDNB concentration and the enzyme-CDNB complex shows a K(i) = 2.7 mM. Concomitant to the inhibition process is formation of an absorption band at 340 nm. After trypsin digestion and HPLC analysis, the CDNB-reacted enzyme gives a single peptide absorbing at 340 nm. Automated Edman degradation of this peptide indicates cysteine 47 to be the residue alkylated by CDNB.
Caccuri
A M
AM
Department of Biology, University of Rome Tor Vergata, Italy.
Petruzzelli
R
R
Polizio
F
F
Federici
G
G
Desideri
A
A
D000595:Amino Acid Sequence; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine; D005260:Female; D005982:Glutathione Transferase / Q000037:antagonists & inhibitors*; D006801:Humans; D007527:Isoenzymes / Q000037:antagonists & inhibitors*; D007700:Kinetics; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000302:isolation & purification; D010920:Placenta / Q000201:enzymology*; D011247:Pregnancy; D014357:Trypsin
D007527:Isoenzymes; D010446:Peptide Fragments; D005982:Glutathione Transferase; D014357:Trypsin; D003545:Cysteine