The importance of a critical protonation state and the fate of the catalytic steps in class A β-lactamases and penicillin-binding proteins
The importance of a critical protonation state and the fate of the catalytic steps in class A β-lactamases and penicillin-binding proteins
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DOI：
10.1074/jbc.m313143200
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发表时间：
2004-08-13
影响因子：
4.8
通讯作者：
Mobashery, S
中科院分区：
文献类型：
--
作者：
Golemi-Kotra, D;Meroueh, SO;Mobashery, S
β-内酰胺酶和青霉素结合蛋白是分别涉及对β-内酰胺抗生素的抗生素抗性和细胞壁的生物合成组装的细菌酶。这些酶大家族的成员都经历了在其活性位点丝氨酸上被其各自底物酰化作为其催化活性的第一步。在所有这些蛋白质中均可见Ser-X-X-Lys序列基序，晶体结构表明丝氨酸和赖氨酸的侧链功能彼此接触。在本报告中使用了三种独立的方法来解决这个重要的赖氨酸（赖氨酸-73）在TEM-1 β-内酰胺酶从大肠杆菌的质子化状态的问题。这些技术包括通过γ-硫代赖氨酸-73变体的研究和伴随的动力学分析来扰动赖氨酸-73的pK（a），通过核磁共振滴定特异性标记的蛋白质来研究质子化状态，以及通过使用热力学积分方法的计算处理。所有三种方法均表明该酶的Lys-73的pK（a）被减弱至8.0 - 8.5。本文认为，A类β-内酰胺酶的Glu-166和Lys-73的独特基态离子对实际上使活性位点赖氨酸的pK（a）从亲本青霉素结合蛋白的pK（a）提高到8.0 - 8.5。虽然我们不能排除Glu-166可能激活活性位点水，这反过来又促进Ser-70的酰化事件，如前面提出的，我们想提出一个合理的替代酰化步骤的可能性，离子对将重新配置为质子化的Glu-166和未质子化的Lys-73。因此，未质子化的Lys-73可以促进丝氨酸酰化，这是一个在所有活性位点丝氨酸β-内酰胺酶和青霉素结合蛋白中共享的过程。
beta-Lactamases and penicillin-binding proteins are bacterial enzymes involved in antibiotic resistance to beta-lactam antibiotics and biosynthetic assembly of cell wall, respectively. Members of these large families of enzymes all experience acylation by their respective substrates at an active site serine as the first step in their catalytic activities. A Ser-X-X-Lys sequence motif is seen in all these proteins, and crystal structures demonstrate that the side-chain functions of the serine and lysine are in contact with one another. Three independent methods were used in this report to address the question of the protonation state of this important lysine (Lys-73) in the TEM-1 beta-lactamase from Escherichia coli. These techniques included perturbation of the pK(a) of Lys-73 by the study of the gamma-thialysine-73 variant and the attendant kinetic analyses, investigation of the protonation state by titration of specifically labeled proteins by nuclear magnetic resonance, and by computational treatment using the thermodynamic integration method. All three methods indicated that the pK(a) of Lys-73 of this enzyme is attenuated to 8.0 - 8.5. It is argued herein that the unique ground-state ion pair of Glu-166 and Lys-73 of class A beta-lactamases has actually raised the pK(a) of the active site lysine to 8.0 - 8.5 from that of the parental penicillin-binding protein. Whereas we cannot rule out that Glu-166 might activate the active site water, which in turn promotes Ser-70 for the acylation event, such as proposed earlier, we would like to propose as a plausible alternative for the acylation step the possibility that the ion pair would reconfigure to the protonated Glu-166 and unprotonated Lys-73. As such, unprotonated Lys-73 could promote serine for acylation, a process that should be shared among all active-site serine beta-lactamases and penicillin-binding proteins.