Zhan-Poe X, Craft C M
Doheny Eye Institute, Department of Cell and Neurobiology, University of Southern California School of Medicine, Los Angeles 90033, USA.
J Pineal Res. 1999 Aug;27(1):49-58. doi: 10.1111/j.1600-079x.1999.tb00596.x.
Pineal and retinal melatonin synthesis is controlled by the enzymatic activity of arylalkylamine N-acetyltransferase (AA-NAT, EC 2.3.1.87), which is regulated by light/dark signals and circadian factors. This enzyme converts serotonin to N-acetylserotonin by the transfer of an acetyl group from acetyl coenzyme A. Endogenous AA-NAT instability during routine purification has made enzyme characterization difficult, but now a stable recombinant protein for AA-NAT has been synthesized to investigate the intrinsic biochemical properties of AA-NAT from a rat pineal cDNA encoding a 205 amino acid, 23 kilodalton protein, by using a glutathione-S-transferase (GST) fusion protein system. Recombinant GST-AA-NAT showed substrate specificity for arylalkylamines and stability at 4 degrees C; however, the enzyme activity was reduced by 40% upon preincubation at 37 degrees C for 2 hr. GST-AA-NAT is preferentially phosphorylated by either cyclic AMP- or cyclic GMP-dependent kinases in vitro, but no detrimental effect was observed on AA-NAT enzymatic activity. Among the metal cations tested in this study, Ca2+, Mg2+, Mn2+, Fe2+, and Co2 showed little or no inhibitory potency, while either 1 mM Zn2+ or 0.1 mM Cu2+ nearly abolished the enzymatic activity. GST-AA-NAT enzyme activity is also inhibited by reagents that are known biochemically to modify thiol groups (N-ethylmaleimide, NEM) and histidine residues (p-chloromercuribenzoate, NBS and diethyl pyrocarbonate, DEPC), suggesting the presence of essential cysteine and histidine moieties. Moreover, preincubation of acetyl CoA completely protects the recombinant AA-NAT from inactivation by NEM and DEPC, indicating that specific cysteine and histidine residues may be at the acetylation site. The conclusion is that the biochemical properties of rat recombinant AA-NAT is similar to the endogenous pineal and retinal AA-NAT with respect to the sensitivity to temperature, metal cations, as well as the thiol modification reagents. These data also suggest that the phosphorylation status of the AA-NAT does not affect enzymatic activity directly, and histidine residues are potentially important residues required for high catalytic activity.
松果体和视网膜褪黑素的合成受芳基烷基胺N - 乙酰基转移酶（AA - NAT，EC 2.3.1.87）的酶活性控制，该酶受光/暗信号和昼夜节律因子调节。这种酶通过将乙酰辅酶A的乙酰基转移，将血清素转化为N - 乙酰血清素。在常规纯化过程中内源性AA - NAT的不稳定性使得酶的特性鉴定变得困难，但现在已经合成了一种稳定的AA - NAT重组蛋白，通过使用谷胱甘肽 - S - 转移酶（GST）融合蛋白系统，从编码205个氨基酸、23千道尔顿蛋白的大鼠松果体cDNA来研究AA - NAT的内在生化特性。重组GST - AA - NAT对芳基烷基胺表现出底物特异性，并且在4℃下稳定；然而，在37℃预孵育2小时后，酶活性降低了40%。GST - AA - NAT在体外优先被环磷酸腺苷（cAMP）或环磷酸鸟苷（cGMP）依赖性激酶磷酸化，但对AA - NAT酶活性未观察到有害影响。在本研究中测试的金属阳离子中，Ca2 +、Mg2 +、Mn2 +、Fe2 +和Co2 +显示出很少或没有抑制效力，而1 mM Zn2 +或0.1 mM Cu2 +几乎完全消除了酶活性。GST - AA - NAT酶活性也受到已知能修饰硫醇基团的试剂（N - 乙基马来酰亚胺，NEM）和组氨酸残基的试剂（对氯汞苯甲酸、N - 溴代琥珀酰亚胺和焦碳酸二乙酯，DEPC）的抑制，这表明存在必需的半胱氨酸和组氨酸部分。此外，乙酰辅酶A的预孵育完全保护重组AA - NAT不被NEM和DEPC灭活，表明特定的半胱氨酸和组氨酸残基可能位于乙酰化位点。结论是，大鼠重组AA - NAT的生化特性在对温度、金属阳离子以及硫醇修饰试剂的敏感性方面与内源性松果体和视网膜AA - NAT相似。这些数据还表明AA - NAT的磷酸化状态不会直接影响酶活性，并且组氨酸残基可能是高催化活性所需的潜在重要残基。
