THE CHARACTERISTICS OF ARYLAMINE N-ACETYLTRANSFERASE IN PSEUDOMONAS-AERUGINOSA

N-Acetyltransferase (NAT), responsible for bioactivation and detoxific ation of arylamines, has been demonstrated to be widely distributed in many organisms ranging from humans to microorganisms. Using high perf ormance liquid chromatography (HPLC) to analyze NAT activity in bacter ia, the authors found that Pseudomonas aeruginosa exhibited high NAT a ctivity with 2-aminofluorene (2-AF) as substrate. Characteristics of t his bacterial NAT were further investigated. The N-acetylation catalyz ed by this enzyme is an acetyl coenzyme A (AcCoA)-dependent reaction. As the concentration of AcCoA in the reaction mixture was increased, t he apparent K-m and V-max for 2-AF increased. The K-m and V-max were 0 .504 +/- 0.056 mM and 31.92 +/- 3.23 nmol/min/mg protein, respectively , for the acetylation of 2-AF with 0.5 mM AcCoA. The optimum pH for th e enzyme activity was estimated to be around 8.5. It was active at a t emperature range from 5 degrees C to 55 degrees C, with maximum activi ty at 37 degrees C. The enzyme activity was inhibited by divalent meta l ions including Cu++, Fe++, Zn++, Ca++, Co++, Mn++, and Mg++, suggest ing that a sulfhydryl group is involved in the N-acetylation activity. The three chemical modification agents, iodoacetamide, phenylglyoxal, and diethylpyrocarbonate, all exhibited a dose-, time-, and temperatu re-dependent inhibition effect. Preincubation of the NAT with AcCoA pr ovided significant protection against the inhibition of iodoacetamide and diethylpyrocarbonate, but only partial protection against the inhi bition of phenylglyoxal. These results indicate that cysteine, histidi ne, and arginine residues are essential for this bacterial enzyme acti vity, and the first two are likely to reside on the AcCoA binding site , but arginine residue may be located only near the AcCoA binding site . Our data demonstrate that P. aeruginosa possesses highly active N-ac etyltransferase which shares a similar catalytic mechanism as that of higher organisms. These findings are very helpful for further investig ating the role of arylamine NAT in this bacterial species.