Hsieh S E, Lo H H, Chung J G
Department of Medical Technology, Chungtai Junior College, Taiwan, Republic of China.
Curr Microbiol. 1998 Jun;36(6):353-60. doi: 10.1007/s002849900322.
N-Acetyltransferase (NAT), responsible for bioactivation and detoxification of arylamines, has been demonstrated to be widely distributed in many organisms ranging from humans to microorganisms. Using high performance liquid chromatography (HPLC) to analyze NAT activity in bacteria, the authors found that Pseudomonas aeruginosa exhibited high NAT activity with 2-aminofluorene (2-AF) as substrate. Characteristics of this bacterial NAT were further investigated. The N-acetylation catalyzed by this enzyme is an acetyl coenzyme A (AcCoA)-dependent reaction. As the concentration of AcCoA in the reaction mixture was increased, the apparent K(m) and Vmax for 2-AF increased. The K(m) and Vmax 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 the enzyme activity was estimated to be around 8.5. It was active at a temperature range from 5 degrees C to 55 degrees C, with maximum activity at 37 degrees C. The enzyme activity was inhibited by divalent metal ions including Cu++, Fe++, Zn++, Ca++, Co++, Mn++, and Mg++, suggesting 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 temperature-dependent inhibition effect. Preincubation of the NAT with AcCoA provided significant protection against the inhibition of iodoacetamide and diethylpyrocarbonate, but only partial protection against the inhibition of phenylglyoxal. These results indicate that cysteine, histidine, and arginine residues are essential for this bacterial enzyme activity, 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-acetyltransferase which shares a similar catalytic mechanism as that of higher organisms. These findings are very helpful for further investigating the role of arylamine NAT in this bacterial species.
N - 乙酰转移酶（NAT）负责芳基胺的生物活化和解毒，已被证明广泛分布于从人类到微生物的许多生物体中。作者使用高效液相色谱法（HPLC）分析细菌中的NAT活性，发现铜绿假单胞菌以2 - 氨基芴（2 - AF）为底物时表现出高NAT活性。对这种细菌NAT的特性进行了进一步研究。该酶催化的N - 乙酰化反应是一种依赖于乙酰辅酶A（AcCoA）的反应。随着反应混合物中AcCoA浓度的增加，2 - AF的表观K(m)和Vmax增加。在0.5 mM AcCoA存在下，2 - AF乙酰化反应的K(m)和Vmax分别为0.504±0.056 mM和31.92±3.23 nmol/min/mg蛋白质。该酶活性的最适pH估计约为8.5。它在5℃至55℃的温度范围内有活性，在37℃时活性最高。该酶活性受到包括Cu++、Fe++、Zn++、Ca++、Co++、Mn++和Mg++在内的二价金属离子的抑制，表明巯基参与了N - 乙酰化活性。三种化学修饰剂碘乙酰胺、苯乙二醛和焦碳酸二乙酯均表现出剂量、时间和温度依赖性抑制作用。NAT与AcCoA预孵育可显著保护其免受碘乙酰胺和焦碳酸二乙酯的抑制，但仅部分保护其免受苯乙二醛的抑制。这些结果表明，半胱氨酸、组氨酸和精氨酸残基对于这种细菌酶的活性至关重要，前两者可能位于AcCoA结合位点，但精氨酸残基可能仅位于AcCoA结合位点附近。我们的数据表明，铜绿假单胞菌拥有高活性的N - 乙酰转移酶，其催化机制与高等生物相似。这些发现对于进一步研究芳基胺NAT在该细菌物种中的作用非常有帮助。
