Substrate specificity, metal binding properties, and spectroscopic characterization of the DapE-encoded N-succinyl-L,L-diaminopimelic acid desuccinylase from Haemophilus influenzae
Substrate specificity, metal binding properties, and spectroscopic characterization of the DapE-encoded N-succinyl-L,L-diaminopimelic acid desuccinylase from Haemophilus influenzae
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DOI：
10.1021/bi034845
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发表时间：
2003-09-16
期刊：
影响因子：
2.9
通讯作者：
Holz, RC
中科院分区：
文献类型：
--
作者：
Bienvenue, DL;Gilner, DM;Holz, RC
研究了流感嗜血杆菌（Haemophilusinfluenzae）DapE编码的N-琥珀酰-L，L-二氨基庚二酸脱琥珀酰酶（DapE）中二价金属离子辅因子结合位点的催化和结构性质。Co（II）取代的DapE酶比Zn（II）负载形式的酶活性高25%。有趣的是，Mn（II）可以激活DapE，但仅能激活20%的Zn（II）负载酶。所观察到的k（cat）值的顺序为Co（II）> Zn（II）> Cd（II）> Mn（II）> Ni（II），与Cu（II）类似，与Mg（II）类似。DapE仅水解L，L-N-琥珀酰-二氨基庚二酸（L，L-SDAP），对D，L-、L，D-和D，D-SDAP无活性。DapE对几种乙酰化氨基酸以及D，L-琥珀酰氨基庚二酸也无活性，其与天然底物L，L-SDAP的不同之处在于氨基酸侧链上不存在antine基团。这些数据意味着琥珀酰基部分的羧酸酯和胺与DapE的活性位点形成重要的相互作用。DapE对一个和两个Zn（II）离子的亲和力相差近2.2 × 10（3）倍（K-d1 = 0.14 μ M vs K-d2 = 300 μ M）。此外，根据在16 ℃和35 ℃之间测量的k（cat）值构建了Arrhenius图，并且在该温度范围内呈线性。发现[ZnZn（DapE）]的活化能为31 kJ/mol，在25 ℃下计算的其余热力学参数为Δ G double dagger = 64 kJ/mol，Δ H double dagger = 28.5 kJ/mol，和Δ S double dagger = -119 J mol（-1）K-1。[Co_（DapE）]和[CoCo（DapE）]的电子吸收和EPR谱表明，Co（II）的第一个结合位是五配位，第二个结合位是八面体。此外，[CoCo（DapE）]中两个Co（II）离子之间的任何自旋-自旋相互作用都非常弱。本文提供的动力学和光谱数据表明，来自H.流感病毒具有类似的二价金属结合特性的氨肽酶从气单胞菌蛋白水解（AAP），和观察到的二价金属离子结合特性进行了讨论，相对于它们在SDAP水解的催化作用。
The catalytic and structural properties of divalent metal ion cofactor binding sites in the dapE-encoded N-succinyl-L,L-diaminopimelic acid desuccinylase (DapE) from Haemophilus influenzae were investigated. Co(II)-substituted DapE enzyme was 25% more active than the Zn(II)-loaded form of the enzyme. Interestingly, Mn(II) can activate DapE, but only to similar to20% of the Zn(II)-loaded enzyme. The order of the observed k(cat) values are Co(II) > Zn(II) > Cd(II) > Mn(II) > Ni(II) similar to Cu(II) similar to Mg(II). DapE was shown to only hydrolyze L,L-N-succinyl-diaminopimelic acid (L,L-SDAP) and was inactive toward D,L-, L,D-, and D,D-SDAP. DapE was also inactive toward several acetylated amino acids as well as D,L-succinyl aminopimelate, which differs from the natural substrate, L,L-SDAP, by the absence of the an-tine group on the amino acid side chain. These data imply that the carboxylate of the succinyl moiety and the amine form important interactions with the active site of DapE. The affinity of DapE for one versus two Zn(II) ions differs by nearly 2.2 x 10(3) times (K-d1 = 0.14 muM vs K-d2 = 300 muM). In addition, an Arrhenius plot was constructed from k(cat) values measured between 16 and 35 degreesC and was linear over this temperature range. The activation energy for [ZnZn(DapE)] was found to be 31 kJ/mol with the remaining thermodynamic parameters calculated at 25 degreesC being DeltaGdouble dagger = 64 kJ/mol, DeltaHdouble dagger = 28.5 kJ/mol, and DeltaSdouble dagger = -119 J mol(-1) K-1. Electronic absorption and EPR spectra of [Co_(DapE)] and [CoCo(DapE)] indicate that the first Co(II) binding site is five-coordinate, while the second site is octahedral. In addition, any spin-spin interaction between the two Co(II) ions in [CoCo(DapE)] is very weak. The kinetic and spectroscopic data presented herein suggest that the DapE from H. influenzae has similar divalent metal binding properties to the aminopeptidase from Aeromonas proteolytica (AAP), and the observed divalent metal ion binding properties are discussed with respect to their catalytic roles in SDAP hydrolysis.