Metal binding Asp-120 in metallo-β-lactamase L1 from Stenotrophomonas maltophilia plays a crucial role in catalysis
Metal binding Asp-120 in metallo-β-lactamase L1 from Stenotrophomonas maltophilia plays a crucial role in catalysis
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DOI：
10.1074/jbc.m309852200
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发表时间：
2004-01-09
影响因子：
4.8
通讯作者：
Crowder, MW
中科院分区：
文献类型：
--
作者：
Garrity, JD;Carenbauer, AL;Crowder, MW
嗜麦芽寡养单胞菌的金属β-内酰胺酶L1是一种双核锌（II）酶，含有一个金属结合天冬氨酸，可能在催化中发挥重要作用。这种金属结合天冬氨酸的存在似乎是常见的大多数双核，含金属，水解酶，特别是那些与β-内酰胺酶折叠。在努力探索的催化和金属结合作用的Asp-120在L1，三个定点突变体（D120 C，D120 N，和D120 S）的制备和表征使用金属分析，圆二色谱，和预稳态和稳态动力学。D120 C、D120 N和D120 S突变体显示分别结合1.6+/-0.2、1.8 +/-0.2和1.1+/-0.2 mol Zn（II）/单体。与野生型L1相比，突变体的k（cat）值下降了10- 1000倍，并且对于所有测试的底物，观察到活性的总体趋势为野生型> D120 N> D120 C和D120 S。溶剂同位素和pH依赖性研究表明，在所有酶的限速步骤期间，一个或多个质子在飞行中，pK（a）值在pH 5-10的范围之外（除了D120 N）。这些数据表明，Asp-120是至关重要的L1结合其完整的锌（II）的补充，并随后适当的底物结合酶。这项工作还证实了Asp-120在催化中起着重要作用，可能是通过与水的氢键结合，帮助形成桥接氢氧化物/水，以及在水解反应中的限速质子转移。
Metallo-beta-lactamase L1 from Stenotrophomonas maltophilia is a dinuclear Zn(II) enzyme that contains a metal-binding aspartic acid in a position to potentially play an important role in catalysis. The presence of this metal-binding aspartic acid appears to be common to most dinuclear, metal-containing, hydrolytic enzymes; particularly those with a beta-lactamase fold. In an effort to probe the catalytic and metal-binding role of Asp-120 in L1, three site-directed mutants (D120C, D120N, and D120S) were prepared and characterized using metal analyses, circular dichroism spectroscopy, and pre-steady-state and steady-state kinetics. The D120C, D120N, and D120S mutants were shown to bind 1.6+/-0.2, 1.8+/-0.2, and 1.1+/-0.2 mol of Zn(II) per monomer, respectively. The mutants exhibited 10- to 1000-fold drops in k(cat) values as compared with wild-type L1, and a general trend of activity, wild-type>D120N>D120C and D120S, was observed for all substrates tested. Solvent isotope and pH dependence studies indicate one or more protons in flight, with pK(a) values outside the range of pH 5-10 (except D120N), during a rate-limiting step for all the enzymes. These data demonstrate that Asp-120 is crucial for L1 to bind its full complement of Zn( II) and subsequently for proper substrate binding to the enzyme. This work also confirms that Asp-120 plays a significant role in catalysis, presumably via hydrogen bonding with water, assisting in formation of the bridging hydroxide/water, and a rate-limiting proton transfer in the hydrolysis reaction.