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

N5-(L-1-羧乙基)-L-鸟氨酸合成酶(CEO， 乳链球菌EC 1.5.1.24)由四个相同的 35,300兆瓦的子机组。这种酶催化一种 丙酮酸与NADPH依赖的还原缩合反应 L-鸟氨酸或L-赖氨酸的侧链氨基，可以是 蛋白质赖氨酸翻译后修饰的重要性 残留物。酶分析、内源性酪氨酸基和色氨基 荧光、硫醇滴定、疏水基团暴露、 光散射、远紫外圆二向色性、二阶导数UV 吸收、尺寸排除层析和分析 采用超速离心法监测细胞变性和细胞毒性。 揭开CEO之路。在0.2M GdnHCl或0.7M氯化钠下，约 观察到1.6倍的酶活性。在约1M GdnHCl， 首席执行官们被停用了。时间、温度和 发生了浓度依赖的可溶性聚集体的形成 在0.5M-1.5M GdnHCl浓度下，由于非共价态 由于非极点表面暴露增加而产生的相互作用。 观察到从四聚体到未折叠单体的转变。 在2-3.5M GdnHCl之间(不含可见的三聚体或二聚体 中间体)，如Tyr、Typ和巯基所证明的 暴露，二级结构的丧失，凝胶过滤洗脱曲线， 和沉积平衡。的解离和展开 四聚体首席执行官们是一致的。重新激活的CEO的收益率 5M GdnHCl的100倍稀释在以下情况下得到改善 解离和重组发生在0度而不是25度。 C，并随着CEO集中度的增加而进一步提高 减少了。未折叠的亚基(30微摩尔)和 从5M GdnHCl在0稀释100倍时的四聚体组装 在以下情况下，C度增加了约4倍(恢复到28%) 与大肠杆菌分子伴侣在15℃下孵育3h GroEL，镁-ATP，100 mM KCl，20 mM Tris，pH 7.2。

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.