McGregor Wade C, Swierczek Sabina I, Bennett Brian, Holz Richard C
Contribution from the Department of Chemistry and Biochemistry, Utah State University, Logan, Utah 84322-0300 and National Biomedical EPR Center, Department of Biophysics, Medical College of Wisconsin, Milwaukee, Wisconsin 53226-0509.
J Am Chem Soc. 2005 Oct 12;127(40):14100-7. doi: 10.1021/ja054081g.
The catalytic and structural properties of the argE-encoded N-acetyl-L-ornithine deacetylase (ArgE) from Escherichia coli were investigated. On the basis of kinetic and ITC (isothermal titration calorimetry) data, Zn(II) binds to ArgE with Kd values that differ by approximately 20 times. Moreover, ArgE exhibits approximately 90% of its full catalytic activity upon addition of one metal ion. Therefore, ArgE behaves similarly to the aminopeptidase from Aeromonas proteolytica (AAP) in that one metal ion is the catalytic metal ion while the second likely plays a structural role. The N-acetyl-L-ornithine (NAO) deacetylase activity of ArgE showed a linear temperature dependence from 20 to 45 degrees C, indicating that the rate-limiting step does not change over this temperature range. The activation energy for NAO hydrolysis by ArgE was 25.6 kJ/mol when loaded with Zn(II) and 34.3 kJ/mol when loaded with Co(II). Electronic absorption and EPR (electron paramagnetic resonance) spectra of [Co x (ArgE)] and [CoCo(ArgE)] indicate that both divalent metal binding sites are five coordinate. In addition, EPR data show clear evidence of spin-spin coupling between the Co(II) ions in the active site but only after addition of a second equivalent of Co(II). Combination of these data provides the first physical evidence that the ArgE from E. coli contains a dinuclear Zn(II) active site, similar to AAP and the carboxypeptidase G2 from Pseudomonas sp. strain RS-16 (CPG2).
对大肠杆菌中由argE编码的N - 乙酰 - L - 鸟氨酸脱乙酰酶（ArgE）的催化和结构特性进行了研究。基于动力学和等温滴定量热法（ITC）数据，锌（II）与ArgE结合的解离常数（Kd）值相差约20倍。此外，添加一个金属离子后，ArgE展现出约90%的最大催化活性。因此，ArgE的行为与解蛋白气单胞菌（Aeromonas proteolytica）的氨肽酶（AAP）相似，即一个金属离子是催化金属离子，而另一个可能起结构作用。ArgE的N - 乙酰 - L - 鸟氨酸（NAO）脱乙酰酶活性在20至45摄氏度范围内呈现线性温度依赖性，这表明在此温度范围内限速步骤没有变化。当负载锌（II）时，ArgE催化NAO水解的活化能为25.6 kJ/mol，负载钴（II）时为34.3 kJ/mol。[Co x (ArgE)]和[CoCo(ArgE)]的电子吸收光谱和电子顺磁共振（EPR）光谱表明，两个二价金属结合位点都是五配位的。此外，EPR数据清楚地表明活性位点中的钴（II）离子之间存在自旋 - 自旋耦合，但这仅在添加第二个当量的钴（II）之后才出现。这些数据的结合提供了首个物理证据，即大肠杆菌的ArgE含有一个双核锌（II）活性位点，类似于AAP和假单胞菌属RS - 16菌株的羧肽酶G2（CPG2）。
