Cloning, expression and mutagenesis of a subunit contact of rabbit muscle-specific (ββ) enolase

Mary Judith Kornblatt , Shu-Xian Zheng , Noel Lamandé , Monique Lazar
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引用次数: 6

Abstract

The cDNA for rabbit muscle-specific (ββ) enolase was cloned, sequenced and expressed in Escherichia coli. This ββ-enolase differs at eight positions from that sequenced by Chin (17). Site-directed mutagenesis was used to change residue 414 from glutamate to leucine, thereby abolishing a salt bridge involved in subunit contacts. Recombinant wild-type and mutant enolase were purified from E. coli and compared to enolase isolated from rabbit muscle. Molecular weights were determined by mass spectrometry. All three ββ-enolases had similar kinetics, and UV and circular dichroism (CD) spectra. The mutant enolase was far more sensitive to inactivation by pressure, by KCl or EDTA, and by sodium perchlorate. We confirmed, by analytical ultracentrifugation, that the sodium perchlorate inactivation was due to dissociation. ΔGo for dissociation of enolase was decreased from 49.7 kJ/mol for the wild-type enzyme to 42.3 kJ/mol for the mutant. In contrast to the wild-type enzyme, perchlorate inactivation of E414L was accompanied by a small loss of secondary structure.

兔肌肉特异性烯醇化酶亚基接触体的克隆、表达和诱变
兔肌肉特异性(ββ)烯醇化酶cDNA克隆、测序并在大肠杆菌中表达。该ββ-烯醇化酶在8个位置上与Chin(17)的测序结果不同。利用定点诱变将残基414从谷氨酸转变为亮氨酸,从而消除了参与亚基接触的盐桥。从大肠杆菌中纯化了重组野生型和突变型烯醇化酶,并与兔肌中分离的烯醇化酶进行了比较。质谱法测定分子量。三种ββ烯醇化酶具有相似的动力学,紫外和圆二色性(CD)光谱。突变体烯醇化酶对压力、KCl或EDTA以及高氯酸钠的失活更为敏感。我们证实,通过分析超离心,高氯酸钠失活是由于解离。烯醇化酶解离速度从野生型酶的49.7 kJ/mol降低到突变体酶的42.3 kJ/mol。与野生型酶相比,E414L的高氯酸盐失活伴随着二级结构的少量损失。
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