Probing the catalytic potential of the hamster arylamine N-acetyltransferase 2 catalytic triad by site-directed mutagenesis of the proximal conserved residue, Tyr190.
Arylamine N-acetyltransferases (NATs) play an important role in both the detoxification of arylamine and hydrazine drugs and the activation of arylamine carcinogens. Because the catalytic triad, Cys-His-Asp, of mammalian NATs has been shown to be essential for maintaining protein stability, rendering it impossible to assess alterations of the triad on catalysis, we explored the impact of the highly conserved proximal residue, Tyr190, which forms a direct hydrogen bond interaction with one of the triad residues, Asp122, as well as a potential pi-pi stacking interaction with the active site His107. The replacement of hamster NAT2 Tyr190 by either Phe, Ile or Ala was well tolerated and did not result in significant alterations in the overall fold of the protein. Nevertheless, stopped-flow and steady-state kinetic analysis revealed that Tyr190 was critical for maximizing the acetylation rate of NAT2 and the transacetylation rate of p-aminobenzoic acid when compared with the wild-type. Tyr190 was also shown to play an important role in determining the pK(a) of the active site Cys during acetylation, as well as the pH versus the rate profile for transacetylation. We hypothesized that the pH dependence was associated with global changes in the active site structure, which was revealed by the superposition of [(1)H, (15)N] heteronuclear single quantum coherence spectra for the wild-type and Y190A. These results suggest that NAT2 catalytic efficiency is partially governed by the ability of Tyr190 to mediate the collective impact of multiple side chains on the electrostatic potential and local conformation of the active site.
Department of Medicinal Chemistry, University of Minnesota, Minneapolis, MN 55455, USA.
Xin
X
Zhou
Naixia
N
Zhang
Li
L
Liu
Kylie J
KJ
Walters
Patrick E
PE
Hanna
Carston R
CR
Wagner
D000818:Animals; D001191:Arylamine N-Acetyltransferase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D002384:Catalysis; D017124:Conserved Sequence; D006224:Cricetinae; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009596:Nitrophenols / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D014443:Tyrosine / Q000737:chemistry* / Q000235:genetics
D009596:Nitrophenols; D014443:Tyrosine; C008642:4-nitrophenyl acetate; D001191:Arylamine N-Acetyltransferase