STRUCTURE OF A PHOSPHONATE-INHIBITED BETA-LACTAMASE - AN ANALOG OF THE TETRAHEDRAL TRANSITION-STATE INTERMEDIATE OF BETA-LACTAM HYDROLYSIS
STRUCTURE OF A PHOSPHONATE-INHIBITED BETA-LACTAMASE - AN ANALOG OF THE TETRAHEDRAL TRANSITION-STATE INTERMEDIATE OF BETA-LACTAM HYDROLYSIS
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DOI：
10.1006/jmbi.1993.1571
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发表时间：
1993-11-05
影响因子：
5.6
通讯作者：
HERZBERG, O
中科院分区：
文献类型：
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作者：
CHEN, CCH;RAHIL, J;HERZBERG, O
测定了金黄色葡萄球菌β-内酰胺酶（β-lactamase）的晶体结构，并在2·3 μ m分辨率下进行了精细化。结构揭示了四面体磷共价键合到活性位点丝氨酸的Oγ原子，Ser 70。与磷键合的氧原子之一位于由Ser 70和Gln 237的两个主链氮原子形成的氧阴离子空穴中，并且第二键合的氧原子被溶剂化。（苄氧基羰基）氨甲基基团朝向活性位点沟，使得肽基团与酶上的极性基团形成补偿静电相互作用。苄基与Ile 239形成疏水相互作用，与Tyr 105形成芳香-芳香边-面相互作用，Tyr 105相对于天然结构经历了构象转变。结合的模式支持的建议，在与酶反应，膦酸酯产生类似的四面体过渡态/与正常底物的酰化步骤相关的中间体的结构。膦酰基在该复合物中的配置与二异丙基磷酰氟抑制胰蛋白酶所产生的复合物中相应的磷酰基的配置相同。该结构与遵循缔合途径的失活机制一致，通过过渡态/中间体进行，其中磷是五配位的，与膦酰基供体（对硝基苯酚）和受体（Ser 70 Oγ原子）在顶端位置形成三角双锥几何结构。这种过渡态的模型可以容纳在β-内酰胺酶的活性位点中，而没有任何空间位阻。本文导出了苄青霉素酰化反应的四面体过渡态模型。由于青霉素分子的稠环的构象刚性，一旦四面体羰基碳及其配体叠加在膦酸酯基团上，底物的取向就被固定。结果是噻唑烷环上的羧酸酯取代基与Lys 234形成盐桥，并且优选的环褶皱是在氨苄青霉素的晶体结构中观察到的，即所谓的“开放”构象异构体。
The crystal structure of β-lactamase fromStaphylococcus aureusinactivated byp-nitrophenyl[[N-(benzyloxycarbonyl)amino]methyl]phosphonate, a methylphosphonate monoester monoanion inhibitor, has been determined and refined at 2·3 Å resolution. The structure reveals a tetrahedral phosphorus covalently bonded to the Oγatom of the active site serine, Ser70. One of the oxygen atoms bonded to phosphorus is located in the oxyanion hole formed by the two main-chain nitrogen atoms of Ser70 and Gln237, and the second bonded oxygen is solvated. The (benzyloxycarbonyl)aminomethyl group is oriented towards the active site gully such that the peptide group forms compensating electrostatic interactions with polar groups on the enzyme. The benzyl group forms a hydrophobic interaction with Ile239 and an aromatic-aromatic edge-to-face interaction with Tyr105, which has undergone a conformational transition relative to the native structure. The mode of binding supports the proposal that on reaction with the enzyme, the phosphonate generates a structure analogous to the tetrahedral transition state/intermediate associated with the acylation step of a normal substrate. The disposition of the phosphonyl group in this complex is the same as that of the corresponding phosphoryl group in the complex resulting from the inhibition of trypsin by diisopropylphosphofluoridate. The structure is consistent with a mechanism of inactivation that follows an associative pathway, proceedingviaa transition state/intermediate in which phosphorus is penta-co-ordinated, forming a trigonal bipyramidal geometry with the phosphonyl donor (p-nitrophenol) and acceptor (Ser70 Oγatom) in apical positions. A model of this transition state can be accommodated in the active site of β-lactamase without any steric hindrance. A model of the tetrahedral transition state associated with the acylation step by benzyl penicillin has been derived. Because of the conformational rigidity of the fused rings of penicillin molecules, the orientation of the substrate is fixed once the tetrahedral carbonyl carbon and its ligands are superimposed on the phosphonate group. The outcome is that the carboxylate substituent on the thiazolidine ring forms a salt bridge with Lys234, and the preferred puckering of the ring is that observed in the crystal structure of ampicillin, the so-called "open" conformer.