Lecture 6

• Glutathione S-transferases (GSTs)

  • Cytosol — mainly soluble enzymes
  • Consists of homo/heterodimers MR = 25,000
  • Reduced glutathione → electrophilic compounds through sulphur/sulfhydryl group
  • Most human tissues
  • Diff isoforms
  • Intracellular binding proteins
• GSTM1 polymorphism


  • Loss of activity
  • Wide range of tissues — reactions incl detoxication of BaP diol epoxide
  • 50% white Europeans lack enzyme
  • 1% Saudi Arabians — duplication of GSTM1
• GSTM1 molecular basis


  1. Homozygous for large deletion

  2. Gene cluster
• GSTM1 analysis


  • Genotyping + phenotyping
  • Activity with trans-stilbene oxide
  • PCR assay
• GSTT1 polymorphism


  • Loss of activity
  • Small organic molecule preference (dichloromethane)
  • 10-20% Caucasians lack enzyme — deletion
• N-acetyltransferases

  • NAT — transfer acetyl group → hydroxyl groups (N-hydroxy group)
  • Both found in cytosol
  • Small proteins products of adjacent intronless genes
  • NAT1 — most tissues, NAT2 — liver + intestine
  • Overlapping substrate specificities
  • Not inducible
• NAT2 polymorphism


  • Isoniazid metabolism
  • 50% Europeans — autosomal recessive
  • Normal activity = fast acetylators
• NAT2 genotyping/phenotyping


  • Phenotype w/ caffeine
  • Genotyping
  • Usually 3 diagnostic reactions
  • Assign genotypes/phenotypes
  • Slow acetylators = *5/*5, *5/*6, *6/*6, *5/*7, *6/*7, *7/*7
• Common NAT2 variant alleles

  • Wild-type = NAT2*4
  • NAT2*5 (C341T (Ile114Thr), C481T, A803G (Lys268Arg))
  • NAT2*6 (C282T, G590A (Arg590Gln))
  • NAT2*7 (G857A (Gly286Glu))
• UDP-glucuronosyltransferases

  • Most used phase II
  • Carried out by UGTs
  • UDP-glucuronic acid — cofactor
  • Endogenous compound conjugated: steroids, vitamins, bile + bilirubin
• UGT genes

  • UGT1A1
  • UGT2B7
• UGT1A1 polymorphism

  • Bilirubin metabolising form
  • Rare inborn errors = Crigler-Najjar syndrome (multiple mutations)
  • Gilbert's syndrome: molecular basis (Higher than normal plasma bilirubin, Most common defect in Caucasians = (TA)7TAA in upstream regulatory region (TATA box) — instead of (TA)6TAA → affects transcription levels, Some Asians — aa sub in exon 1 = same phenotype, Relevant to irinotecan metabolism — anticancer drug)
• UGT2B7 polymorphism


  • Metabolises — morphine + various NSAIDs
  • Common polymorphism = C801T → close to promoter region (C = 1, T=*2)
  • TT = incr morphine 6-glucuronidation in vivo
• SULT1A1 polymorphism


  • Most studied
  • Copy no. variation
  • 3'-UTR involving regulation by miRNA (Section of mRNA following stop codon, Contains elements reg. expression post-translationally, Binds reg. proteins + miRNA, miRNA binding = reduce gene expression → inhibit translation/mRNA cleavage, Effect on SULT1A1 activity (Decr. enzyme activity in homo/heterozygotes for T allele, C973T = 3'end of UTR, More effective miRNA-631 binding = more down-regulation of expression — confirmed with reporter gene assay, 20% white US pop. homozygous)
• Thiopurine methyltransferase (TPMT)


  • Methyltransferases → methylates 6-mercaptopurine (anticancer drug) at thiol group
  • S-adenosylmethionine (SAM) donates methyl group
  • 0.3% Caucasians lack enzyme
• TPMT polymorphism


  • Main variant — aa substitution
  • *2 (G238C → Ala80Pro)
  • *3 (Combinations of G460A (Ala143Thr) (3B) + A719G (Tyr240Cys) (3C) (3A — both))
• Catechol O-methyltransferase (COMT)


  • Metabolism of NA → Normetanephrine
  • Also L-DOPA
• COMT polymorphisms

  • Codon 158 G→A (Val→Met)
  • Met form more thermolabile than Val + lower activity