Biochemical characterization of recombinant serotonin N-acetyltransferase.
Biochemical characterization of recombinant serotonin N-acetyltransferase.
复制标题
重组血清素 N-乙酰转移酶的生化特征。
DOI：
10.1111/j.1600-079x.1999.tb00596.x
复制
发表时间：
1999
影响因子：
10.3
通讯作者：
Craft,CM
中科院分区：
文献类型：
--
作者：
Zhan-Poe,X;Craft,CM
松果体和视网膜褪黑激素的合成受芳烷基胺N-乙酰转移酶（AA-NAT，EC 2）的酶活性控制。3. 1. 87），这是由光/暗信号和昼夜因素调节。这种酶通过转移乙酰辅酶A的乙酰基将血清素转化为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表征变得困难，但是现在已经合成了AA-NAT的稳定重组蛋白，以研究来自大鼠松果体cDNA编码205个氨基酸的AA-NAT的内在生物化学性质。23千道尔顿蛋白，通过使用谷胱甘肽-S-转移酶（GST）融合蛋白系统。重组GST-AA-NAT显示出对芳基烷基胺的底物特异性和在4°C下的稳定性;然而，在37°C下预孵育2小时后，酶活性降低了40%。GST-AA-NAT在体外优先被环AMP-或环GMP-依赖性激酶磷酸化，但未观察到对AA-NAT酶活性的不利影响。在本研究中测试的金属阳离子中，Ca 2+、Mn 2+、Mn 2+、Fe 2-和Co2+显示很少或没有抑制效力，而1 mM Zn 2+或0.1 mM Cu 2+几乎消除酶活性。GST-AA-NAT酶活性也受到已知在生物化学上修饰巯基（N-乙基马来酰亚胺，NEM）和组氨酸残基（对氯汞苯甲酸盐，NBS和焦碳酸二乙酯，DEPC）的试剂的抑制，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免受NEM和DEPC的失活，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的生化性质与内源性松果体和视网膜AA-NAT在对温度、金属阳离子以及巯基修饰试剂的敏感性方面相似。这些数据还表明，AA-NAT的磷酸化状态不会直接影响酶活性，组氨酸残基是高催化活性所需的潜在重要残基。
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°C; however, the enzyme activity was reduced by 40% upon preincubation at 37°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+, Mn2+, 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.