Purification and characterization of guinea-pig liver microsomal deacetylase involved in the deacetylation of the O-glucoside of N-hydroxyacetanilide.
A microsomal deacetylase that catalyses the deacetylation of the O-glucoside of N-hydroxyacetanilide (GHA) was purified from guinea-pig liver. The activity was located exclusively in the microsomes and not detected in the cytosol. The purified GHA deacetylase was a trimeric protein with a molecular mass of 160+/-10 (S.D.) kDa composed of subunits of 53+/-2 kDa; its pI was 4.7. The N-terminal amino acid sequence of GHA deacetylase was similar to those reported for guinea-pig and rat liver microsomal carboxylesterases. The GHA deacetylase showed a comparable hydrolytic activity towards p-nitrophenyl acetate (PNPA), although the activities towards N-hydroxyacetanilide, acetanilide and some endogenous acylated compounds were very low or not detectable. The deacetylase activity towards GHA was inhibited by organophosphates but not by p-chloromercuribenzoate, suggesting that GHA deacetylase can be classified as a B-esterase. The enzyme exhibited a positive homotropic co-operativity towards GHA. The values of the Hill coefficient, the half-saturating concentration ([S]0.5) for GHA, and Vmax were 1.59+/-0.03, 5.51+/-0.07 mM and 32.5+/-1.4 micromol/min per mg respectively, at the optimum pH of 8.5. The bell-shaped pH dependence of the Vmax/[S]0.5 profile indicated pKa values attributed to histidine and lysine residues. The study of stoichiometric inhibition by di-isopropyl fluorophosphate and kinetic analysis with the Monod-Wyman-Changeux model suggests that GHA deacetylase has six substrate binding sites and three catalytically essential serine residues per enzyme molecule.
Suzuki-Kurasaki
M
M
Department of Chemical Hygiene, Hokkaido Institute of Pharmaceutical Sciences, Otaru, 047-02, Japan.
Yoshioka
T
T
Uematsu
T
T
D000117:Acetylglucosamine / Q000031:analogs & derivatives* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D043182:Carboxylesterase; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D002458:Cell Fractionation; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D006168:Guinea Pigs; D007700:Kinetics; D046911:Macromolecular Substances; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D051381:Rats; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
C107088:1-((N-acetyl-N-phenylamino)oxy)-1-deoxy-glucopyranoside; D046911:Macromolecular Substances; D010446:Peptide Fragments; D002265:Carboxylic Ester Hydrolases; D043182:Carboxylesterase; D000117:Acetylglucosamine