Role of arginine 216 in catalytic activity of murine Alpha class glutathione transferases mGSTAl-1 and mGSTA2-2 toward carcinogenic diol epoxides of polycyclic aromatic hydrocarbons.
Murine class Alpha glutathione (GSH) transferase A1-1 (mGSTA1-1) is unique among mammalian Alpha class GSTs due to its exceptionally high catalytic activity toward (+)-anti-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(+)-anti-BPDE], which is the activated metabolite of an environmentally relevant carcinogen, benzo[a] pyrene (BP). However, the molecular basis for high catalytic activity of mGSTA1-1 toward (+)-anti-BPDE is not clear. In the present study, we demonstrate that an arginine residue at position 216, which is conserved in some but not all mammalian class Alpha GSTs, plays an important role in catalytic activity of mGSTA1-1 toward (+)-anti-BPDE and carcinogenic diol epoxides of other environmentally relevant polycyclic aromatic hydrocarbons (PAHs). The catalytic efficiency (k(cat)/K(m)) of mGSTA1-1 for the GSH conjugation of (+)-anti-BPDE (108/mM/s) was reduced by about 58% upon replacement of arginine 216 with alanine (R216A). This was mainly due to a significantly lower V(max) for the R216A mutant of mGSTA1-1 compared with wild-type mGSTA1-1. The R216A mutation also resulted in a statistically significant reduction (>70%) in specific activity of mGSTA1-1 toward racemic anti-diol epoxides of chrysene and benzo[c]phenanthrene (anti-CDE and anti-B[c]PDE, respectively). The catalytic activity of mGSTA2-2, which is a close structural homologue of mGSTA1-1, was also reduced upon R216A mutation. The results of the present study clearly indicate that an arginine residue at position 216 is critical for catalytic activity of mGSTA1-1 and mGSTA2-2 toward carcinogenic diol epoxide metabolites of various PAHs that are abundant in the environment and suspected human carcinogens.
Pal
A
A
Department of Pharmacology and University of Pittsburgh Cancer Institute, University of Pittsburgh School of Medicine, 3550 Terrace Street, Pittsburgh, PA 15261, USA.
Gu
Y
Y
Herzog
C
C
Srivastava
S K
SK
Zimniak
P
P
Ji
X
X
Singh
S V
SV
D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide / Q000378:metabolism*; D000818:Animals; D001120:Arginine / Q000378:metabolism*; D001483:Base Sequence; D002273:Carcinogens / Q000378:metabolism*; D002384:Catalysis; D017931:DNA Primers; D005982:Glutathione Transferase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011083:Polycyclic Compounds / Q000378:metabolism*
D002273:Carcinogens; D017931:DNA Primers; D007527:Isoenzymes; D011083:Polycyclic Compounds; D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide; D001120:Arginine; D005982:Glutathione Transferase