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首页> 外文期刊>Biochemistry >Hydrolysis of N-succinyl-L,L-diaminopimelic acid by the Haemophilus influenzae dapE-encoded desuccinylase: Metal activation, solvent isotope effects, and kinetic mechanism
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Hydrolysis of N-succinyl-L,L-diaminopimelic acid by the Haemophilus influenzae dapE-encoded desuccinylase: Metal activation, solvent isotope effects, and kinetic mechanism

机译：流感嗜血杆菌dapE编码的脱琥珀酰酶水解N-琥珀酰-L，L-二氨基庚二酸：金属活化，溶剂同位素效应和动力学机理

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摘要

Hydrolysis of N-succinyl-L,L-diaminopimelic acid by the dapE-encoded desuccinylase is required for the bacterial synthesis of lysine and meso-diaminopimelic acid, We have investigated the catalytic mechanism of the recombinant enzyme from Haemophilus influenzae. The desuccinylase was overexpressed in Escherichia coli and purified to homogeneity. Steady-state kinetic experiments verified that the enzyme is metal-dependent, with a K-m, for N-succinyl-L,L-diaminopimelic acid of 1.3 mM and a turnover number of 200 s(-1) in the presence of zinc. The maximal velocity was independent of pH above 7 bur decreased with a slope of 1 below pH 7. The pH dependence of V/K was bell-shaped with apparent pKs of 6.5 and 8.3. Both L,L- and D,L-diaminopimelic acid were competitive inhibitors of the substrate, but D,D-diaminopimelic acid was not. Solvent kinetic isotope effect studies yielded inverse isotope effects, with values for V-D2O/K of 0.62 and V-D2O of 0.78. Determination of metal stoichiometry by ICP-AES indicated one tightly bound metal ion, while sequence homologies suggest the presence of two metal binding sites. On the basis of these observations, we propose a chemical mechanism for this metalloenzyme, which has a number of important structurally defined homologues. [References: 44]
机译：dapE编码的脱琥珀酰酶水解N-琥珀酰-L，L-二氨基庚二酸是细菌赖氨酸和内消旋二氨基庚二酸的细菌合成，我们研究了流感嗜血杆菌重组酶的催化机理。脱琥珀酰酶在大肠杆菌中过表达并纯化至同质。稳态动力学实验证明，该酶是金属依赖性的，对于K-m，N-琥珀酰-L，L-二氨基庚二酸为1.3 mM，在锌存在下的转换数为200 s（-1）。最大速度与高于7 bur的pH无关，以低于pH 7的斜率下降1。V/ K的pH依赖性呈钟形，表观pKs为6.5和8.3。 L，L-和D，L-二氨基庚二酸都是底物的竞争性抑制剂，但D，D-二氨基庚二酸不是。溶剂动力学同位素效应研究产生了反同位素效应，V-D2O / K值为0.62，V-D2O值为0.78。 ICP-AES测定金属化学计量表明一个紧密结合的金属离子，而序列同源性表明存在两个金属结合位点。基于这些观察，我们提出了这种金属酶的化学机制，该金属酶具有许多重要的结构上定义的同系物。 [参考：44]

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