TETRAMERIC N5-(CARBOXYETHYL)ORNITHINE SYNTHASE: UNFOLDING AND REFOLDING

四聚体 N5-(羧乙基)鸟氨酸合成酶：解折叠和重折叠

• 批准号: 6290365
• 负责人: ANN GINSBURG
• 金额: --
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6290365
• 关键词: amine oxidoreductase; circular dichroism; enzyme activity; enzyme structure; fluorescence spectrometry; guanidines; oxidoreductase inhibitor; protein folding; ultraviolet spectrometry; urea

Tetrameric N5-(L-1-carboxyethyl)-L-ornithine synthase (CEOS; 141,200 MW) from Lactococcus lactis catalyzes a NADPH-dependent reductive condensation between pyruvate and the side-chain amino group of L- ornithine or L-lysine and may be important for post-translational modification of protein lysyl residues. Guanidine-HCl (GdnHCl)-induced unfolding of this tetrameric enzyme at pH 7.2 and 25 C occurred in several phases. The enzyme was inactivated at ca. 1 M GdnHCl. A time-, temperature-, and concentration-dependent formation of soluble protein aggregates occurred at 0.5-1.5 M GdnHCl due to an increased exposure of apolar surfaces. A transition from tetramer to unfolded monomer was observed between 2 and 3.5 M GdnHCl (without observable dimer or trimer intermediates), as evidenced by tyrosyl and tryptophanyl fluorescence changes, sulfhydryl group exposure, loss of secondary structure, size exclusion chromatography, and sedimentation equilibrium data. GdnHCl- induced dissociation and unfolding of tetrameric CEOS was concerted, and yields of reactivated CEOS by dilution from 5 M GdnHCl were improved when unfolding took place on ice rather than at 25 C. Refolding and reconstitution of the enzyme were optimal at ca. 15 C and yields of active tetramer increased as the protein concentration decreased. Refolding of unfolded subunits and active tetramer assembly upon 100-fold dilution from 5 M GdnHCl on ice also was increased 2- or 4-fold (to 44 or 28 per cent reactivation for 0.08 or 0.28 micromolar subunit, respectively) when incubated at 15 C, pH 7.2 for 4 h with the E. coli molecular chaperonin GroEL, ATP, Mg(II), and KCl. The unusual low-temperature requirement for refolding CEOS results from competing aggregation reactions that become dominant at temperatures higher than 15 C. - N5-(carboxyethyl)ornithine synthase, guanidine hydrochloride, inactivation, tetramer dissociation, unfolding, refolding, GroEL-Mg- ATP, chaperonin-60

来自乳酸乳球菌的四聚体N5-（L-1-羧乙基）-L-鸟氨酸合酶（CEOS; 141，200 MW）催化丙酮酸盐与L-鸟氨酸或L-赖氨酸的侧链氨基之间的NADPH依赖性还原缩合，并且对于蛋白质赖氨酰残基的翻译后修饰可能是重要的。盐酸胍（GdnHCl）诱导的这种四聚体酶在pH 7.2和25 ℃下的解折叠发生在几个阶段。酶在约20 ℃失活。1 M盐酸钆。时间，温度和浓度依赖性的可溶性蛋白质聚集体的形成发生在0.5-1.5 M盐酸钆由于非极性表面的暴露增加。从四聚体到未折叠的单体之间观察到2和3.5 M盐酸钆（没有可观察到的二聚体或三聚体中间体）的过渡，证明酪氨酰和dichanyl荧光变化，巯基暴露，损失的二级结构，尺寸排阻色谱法，和沉降平衡数据。GdnHCl诱导的四聚体CEOS的解离和解折叠是一致的，并且当解折叠在冰上而不是在25 ℃下进行时，通过从5 MGdnHCl中稀释再活化的CEOS的产率得到提高.酶的再折叠和重建在约为最佳。随着蛋白质浓度的降低，活性四聚体的产量增加。当在15 ℃，pH7.2下与E. coli分子伴侣GroEL、ATP、Mg（II）和KCl。重折叠CEOS的不寻常的低温要求是由竞争性聚集反应引起的，这些反应在高于15 ℃的温度下变得占主导地位。- N5-（羧乙基）鸟氨酸合酶，盐酸胍，失活，四聚体解离，去折叠，重折叠，GroEL-Mg- ATP，伴侣蛋白-60