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

来自乳酸乳球菌的四聚体N5-(L-1-羧乙基)-L-鸟氨酸合成酶(CEOS；141,200 mW)催化丙酮酸与L-鸟氨酸或L-赖氨酸侧链氨基之间依赖于NADPH的还原缩合反应，这可能对蛋白质赖氨酸残基的翻译后修饰起重要作用。在pH 7.2和25℃条件下，GdnHCl(GdnHCl)诱导该四聚体酶分几个阶段去折叠。该酶在约1M GdnHCl时失活。在0.5-1.5M的GdnHCl中，由于非极层表面暴露的增加，出现了依赖于时间、温度和浓度的可溶性蛋白质聚集体的形成。在2-3.5M GdnHCl(没有可观察到的二聚体或三聚体中间体)之间观察到从四聚体到未折叠单体的转变，从酪氨酰和色氨酸的荧光变化、巯基暴露、二级结构的损失、尺寸排斥层析和沉淀平衡数据证明了这一点。GdnHCl诱导的四聚体CEOs的解离和去折叠是一致的，在冰上而不是在25℃下，用5M GdnHCl稀释再激活的CEO的产率比在25℃下更高，在15℃左右酶的复性和重组最好，活性四聚体的产率随着蛋白质浓度的降低而增加。当在15℃、pH 7.2条件下与大肠杆菌分子伴侣GroEL、ATP、Mg(II)和KCl孵育4h时，未折叠亚基的复性和活性四聚体组装从5M GdnHCl在冰上100倍稀释时也增加了2倍或4倍(0.08或0.28微摩尔亚基的复活率分别为44%或28%)。对折叠CEO的异常低温要求来自于竞争聚集反应，这些反应在高于15℃的温度下变得主导-N5-(羧乙基)鸟氨酸合成酶、盐酸胍、灭活、四聚体解离、去折叠、复性、GroEL-mg-ATP、伴侣-60

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