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首页> 外文期刊>Biochemistry >Structural Basis for the Altered Activity of Gly794 Variants of Escherichia coli beta-Galactosidase( dagger ).
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Structural Basis for the Altered Activity of Gly794 Variants of Escherichia coli beta-Galactosidase( dagger ).

机译：大肠杆菌β-半乳糖苷酶（dagger）Gly794变体活性改变的结构基础。

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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-beta-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.
机译：通过X射线晶体学和酶动力学研究了大肠杆菌β-半乳糖苷酶活性位点中的开-闭构象转换。丙氨酸替代Gly794导致脱辅酶采取封闭而不是开放的构象。竞争性抑制剂异丙基硫代β-d-半乳糖苷（IPTG）的结合要求突变酶采取其较不受欢迎的开放构象，从而相对于野生型减弱亲和力。相反，过渡态抑制剂以封闭构象结合酶，这对于突变体是有利的，并且相对于野生型显示出增加的亲和力。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 kcal / mol。通过有利于闭合构象，取代相对于天然酶沿着反应坐标移动酶的静止状态，并且相对于第一过渡状态使基态不稳定。结果是半乳糖基化的速率常数增加，但半乳糖基化较慢。共价中间体可以比第二过渡态更好地稳定。取代还导致葡萄糖更好地与游离酶和半乳糖基化酶结合。然而，与天然酶相比，突变体用葡萄糖进行转半乳糖基化以产生异乳糖（lac操纵子的诱导剂）的速度较慢。这表明葡萄糖对于反应而言是错位的，或者与葡萄糖结合的半乳糖基化酶相对于转半乳糖基化的过渡态是稳定的。

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