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Effect of group-selective modification reagents on arylamine N-acetyltransferase activities
Abstract:
Two forms of hamster hepatic arylamine N-acetyltransferase (NAT; EC 2.3.1.5), designated NAT I and NAT II, were purified 200- to 300-fold by sequential 35-50% ammonium sulfate fractionation, Sephadex G-100 gel filtration chromatography, AAB affinity chromatography, DEAE ion exchange chromatography, and P-200 gel filtration chromatography. Treatment of either NAT I or NAT II with N-ethylmaleimide (NEM), a cysteine selective reagent, caused a concentration-dependent loss of enzymatic activities. Acetyl coenzyme A (AcCoA) protected NAT I against inactivation by NEM, whereas both 2-acetylaminofluorene (2-AAF) and AcCoA protected NAT II against inactivation. Incubation of either NAT I or NAT II with phenylglyoxal (PG), an arginine selective reagent, caused a time-dependent and a concentration-dependent loss of both NAT I and NAT II activities; the inactivations followed pseudo first-order kinetics. The reaction order with respect to PG was approximately two for each enzyme, consistent with the expected stoichiometry for the reaction of PG with arginine. The presence of AcCoA provided full protection of NAT I against inactivation by PG. However, neither AcCoA nor 2-AAF provided protection of NAT II against inactivation by PG. Diethylpyrocarbonate (DEPC), a histidine selective reagent, caused time-dependent and concentration-dependent pseudo first-order inactivation of both NAT I and NAT II. Neither AcCoA nor products of NAT-catalyzed reactions protected NAT I and NAT II against inactivation by DEPC. These results suggest that cysteine, arginine and histidine residues are essential to the catalytic activity of both NAT I and NAT II; the cysteine(s) is located at or near the binding site of NAT I and NAT II, and the arginine residue appears to be located in the AcCoA binding site of NAT I. In contrast, the essential arginine residue(s) of NAT II and the essential histidine residue(s) of both NAT I and NAT II are not likely to reside in the binding site of the enzymes.