Glutathione Stransferases:

1

• Glutathione S-transferases
• Multiple forms of the enzyme
• At least 14 classes a, b, d, k, l, m, w, j, p,
  s, q, t, z, and microsomal.
• Many reactions are catalyzed

2

• Glutathione S-transferase reactions
• Almost any lipophilic compound with an
  electrophilic atom

• Attack on N
• Organic nitrate esters

• Attack on C
• S-alkyltransferase
• S-aryltransferase
• S-epoxidetransferase
• S-alkenetransferase
• Alkene isomerisation

• Attack on S
• Organic thiocyanates
• disulfides

• Attack on O
• Organic peroxides

3

• Glutathione S-transferases

4

• Glutathione S-transferase reactions
  dehalogenations

5

• Multistep detoxification with P450, GST,
  hydrolases, and acetyltransferases
• GSH conjugates are often procesed further
• Cleavage of the g-glutamyl moiety by
  g-glutamyl-transpeptidase
• Cleavage of the glycine moiety by cysteinyl
  glycinase
• Acetylation of the cysteine amino group by an
  N-acetyl transferase to form a mercapturic acid
  derivative, that can be excreted

6

• GST, mechanism of action
• Reaction with 1-chloro-2,4-dinitrobenzene (CDNB)
  via 1-chloro-1-(S-glutathionyl)-2,4-dinitrocyclohe
  xadienate (GSCDSC-) to 1-(S-glutathionyl)-2,4-dini
  trobenzene (GSDNB).

Meisenheimer complex
7

• GST, mechanism of action
• Formation of a stable Meisenheimer complex
  (1-(S-glutathionyl)-2,4,6-trinitrocyclohexadienate
  , GSTCD-) is possible with the inhibitor
  trinitrobenzene (TNB) hydride is a poor leaving
  group and three nitro groups are stabilizing.

Meisenheimer complex
8

• GST
• Crystals with intermediates (inhibitor) and
  substrates

GSH GSTCD-
GSDNB substrate inhibitor
product
9

• GST, mechanism of action
• Attack of the nucleophile GS-. The pKa of bound
  glutathione is ca. 2 units lower than in solution
  (8-9). The thiolate is stabilized by hydrogen
  bonding to a Tyr (class mu, pi) or Ser.
  Additionally the helix dipole of the a1-helix
  points to the thiolate.

10

• GST, various residues involved in thiolate
  activation (divergent evolution)
• Theta serine
• Mu tyrosine
• Alpha tyrosine and arginine
• The metal dependent fosfomycin resistance protein
  bears no sequence relationship, but also GSH
  transferase activity
• Convergent evolution.

11

• GST, mechanism of action
• Stabilization of the intermediate In the
  isolated Meisenheimer complex the nitro groups
  are coplanar with the ring (mesomeric forms with
  double bond!), in the enzyme the ortho nitro
  group is rotated out of plane (hydrogen bonds).

12

• GST, mechanism of action
• 1 Red inhibitor GSTCD- (middle NO2 group looking
  to the front) and green H2O at the leaving Cl-
  position.
• 2. Yellow product GSDNB

13

• GST, mechanism of action The product complex

After elimination of the chloride the benzene
ring rotates to the right The p-nitro group
undergoes a movement of almost 8 A during the
reaction, the o-nitro group 6 A (----gt)!
GSDNB
GSTCD-
14

• Multistep detoxification with P450, GST,
  hydrolases, and acetyltransferases

15

• Dehydroascorbate reductase (DHAR) is a GST with
  Cys20 instead of Ser. The GST template has even
  been utilized for a chloride channel

Mechanism with thiohemiketal Single
electron transfer mechanism (SET)