Structural basis for the altered activity of Gly794 variants of Escherichia coli β-galactosidase
Structural basis for the altered activity of Gly794 variants of Escherichia coli β-galactosidase
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DOI：
10.1021/bi035506j
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发表时间：
2003-11-25
期刊：
影响因子：
2.9
通讯作者：
Huber, RE
中科院分区：
文献类型：
--
作者：
Juers, DH;Hakda, S;Huber, RE
用X射线结晶学和酶动力学方法研究了大肠杆菌β-半乳糖苷酶活性部位的开闭构象开关。丙氨酸取代Gly794导致酶采用闭合构象而不是开放构象。竞争性抑制剂异丙基硫代-对-D-半乳糖苷(IPTG)的结合要求突变酶采用其不太受欢迎的开放构象，从而削弱了相对于野生型的亲和力。相反，过渡态抑制剂以封闭的构象与酶结合，这对突变体有利，并显示出相对于野生型更高的亲和力。亲和力的变化表明，封闭形式和开放形式之间的自由能差为1-2千卡/摩尔。通过有利于闭合构象，取代沿着相对于天然酶的反应坐标移动酶的静止状态，并相对于第一过渡态破坏基态的稳定性。结果表明，半乳糖化反应速率常数增大，去乳糖化反应速度变慢。共价中间体可能比第二过渡态更稳定。取代还导致葡萄糖与游离酶和半乳糖化酶更好地结合。然而，与葡萄糖的转半乳糖化以产生别乳糖(lac操纵子的诱导剂)的突变体比用天然酶慢。这表明要么是葡萄糖在反应中错位，要么是与葡萄糖结合的半乳糖化酶相对于转半乳糖化的过渡态是稳定的。
The open-closed conformational switch in the active site of Escherichia coli beta-galactosidase was studied by X-ray crystallography and enzyme kinetics. Replacement of Gly794 by alanine causes the apoenzyme to adopt the closed rather than the open conformation. Binding of the competitive inhibitor isopropyl thio-p-D-galactoside (IPTG) requires the mutant enzyme to adopt its less favored open conformation, weakening affinity relative to wild type. In contrast, transition-state inhibitors bind to the enzyme in the closed conformation, which is favored for the mutant, and display increased affinity relative to wild type. Changes in affinity suggest that the free energy difference between the closed and open forms is 1-2 kcal/mol. By favoring the closed conformation, the substitution moves the resting state of the enzyme along the reaction coordinate relative to the native enzyme and destabilizes the ground state relative to the first transition state. The result is that the rate constant for galactosylation is increased but degalactosylation is slower. The covalent intermediate may be better stabilized than the second transition state. The substitution also results in better binding of glucose to both the free and the galactosylated enzyme. However, transgalactosylation with glucose to produce allolactose (the inducer of the lac operon) is slower with the mutant than with the native enzyme. This suggests either that the glucose is misaligned for the reaction or that the galactosylated enzyme with glucose bound is stabilized relative to the transition state for transgalactosylation.