Tetrameric N5-(Carboxyethyl)ornithine synthase: unfolding and refolding

四聚体 N5-(羧乙基)鸟氨酸合酶：展开和重折叠

• 批准号: 6109159
• 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/6109159
• 关键词: amine oxidoreductase; circular dichroism; enzyme activity; enzyme structure; fluorescence spectrometry; guanidines; oxidoreductase inhibitor; protein folding; ultraviolet spectrometry; urea

N5-(L-1-carboxyethyl)-L-ornithine synthase (CEOS, EC 1.5.1.24) from Lactococcus lactis consists of four identical subunits of 35,300 MW. The enzyme catalyses an 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. Enzyme assays, intrinsic tyrosyl and tryptophanyl fluorescence, thiol group titration, hydrophobic group exposure, light scattering, far-UV circular dichroism, second-derivative UV absorption, size-exclusion chromatography and analytical ultracentrifugation were employed to monitor the denaturation and unfolding pathway of CEOS. At 0.2 M GdnHCl or 0.7 M NaCl, ca 1.6-fold enzyme activation was observed. At ca 1 M GdnHCl, CEOS was inactivated. A time-, temperature-, and concentration-dependent formation of soluble aggregates occurred at 0.5 M - 1.5 M GdnHCl concentrations due to noncovalent interactions arising from an increased exposure of apolar surfaces. A transition from tetramer to unfolded monomers was observed between 2 and 3.5 M GdnHCl (without observable trimer or dimer intermediates), as evidenced by Tyr, Typ, and sulfhydryl group exposure, loss of secondary structure, gel filtration elution profiles, and sedimentation equilibrium. Dissociation and unfolding of tetrameric CEOS was concerted. Yields of reactivated CEOS by 100-fold dilution from 5 M GdnHCl were improved when dissociation and reconstitution took place at 0 rather than at 25 deg C, and were further improved as the concentration of CEOS was decreased. Refolding of unfolded subunits (30 micromolar) and tetramer assembly upon 100-fold dilution from 5 M GdnHCl at 0 deg C was increased ca 4-fold (to 28% reactivation) when incubated at 15 deg C for 3 h with the E. coli molecular chaperonin GroEL, Mg-ATP, 100 mM KCl, and 20 mM Tris, pH 7.2.

N5-（L-1-羧乙基）-L-鸟氨酸合酶（CEOS， EC 1.5.1.24）由四个相同的 分子量为35,300的亚基。这种酶催化 丙酮酸和葡萄糖之间的NADPH依赖性还原缩合 L-鸟氨酸或L-赖氨酸侧链氨基，可以是 赖氨酰对蛋白质翻译后修饰重要性 残基酶测定，内在酪氨酰和酪氨酰 荧光，巯基滴定，疏水基团暴露， 光散射，远紫外圆二色性，二阶导数紫外 吸附、尺寸排阻色谱和分析 采用超离心法监测变性， 地球观测卫星委员会的展开路径。在0.2 M盐酸钆或0.7 M氯化钠，约 1.6-观察到酶活化倍数。在约1 M盐酸钆时， CEOS失活。时间、温度和 可溶性聚集体的形成呈浓度依赖性 在0.5 M - 1.5 M GdnHCl浓度下，由于非共价键 非极性表面暴露增加引起的相互作用。 观察到从四聚体到未折叠单体的转变 2 - 3.5 M GdnHCl（无可观察到的三聚体或二聚体 中间体），如Tyr、Typ和巯基所证明 暴露、二级结构损失、凝胶过滤洗脱图谱， 和沉降平衡。分离和展开 四聚体CEOS是一致的。重新激活的地球观测卫星收益率， 100-5 M GdnHCl的稀释倍数得到改善， 解离和重构发生在0 ℃而不是25 ℃ 随着CEOS浓度的增加， 降低未折叠亚基的重折叠（30微摩尔）和 在0 ℃下从5 M GdnHCl稀释100倍后的四聚体组装 摄氏度增加约4倍（至28%再激活）， 在15 ℃下与E.大肠杆菌分子伴侣蛋白 GroEL、Mg-ATP、100 mM KCl和20 mM Tris，pH 7.2。