PURIFICATION AND PROPERTIES OF THE LIPOATE PROTEIN LIGASE OF ESCHERICHIA-COLI
PURIFICATION AND PROPERTIES OF THE LIPOATE PROTEIN LIGASE OF ESCHERICHIA-COLI
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DOI：
10.1042/bj3090853
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发表时间：
1995-08-01
影响因子：
4.1
通讯作者：
GUEST, JR
中科院分区：
文献类型：
--
作者：
GREEN, DE;MORRIS, TW;GUEST, JR
Lipoate是大肠杆菌2-酮酸脱氢酶复合物和甘氨酸裂解系统的重要组成部分。通过Mg 2+和ATP依赖性硫辛酸蛋白连接酶（LPL）将其连接到丙酮酸脱氢酶复合物E2 p（硫辛酸乙酰转移酶）亚基的硫辛酰结构域中的特定赖氨酸残基上。从野生型E.大肠杆菌中，其丰度极低（每个细胞< 10个分子），并且来自基因扩增来源。纯化的酶是一种单体蛋白（M（r）38 000），形成不规则的针状晶体簇。它在-20 ℃下稳定，但在4 ℃下缓慢氧化为含有至少一个分子内二硫键的无活性形式。非活性形式可通过还原剂或在纯化的最后阶段从LPL中分离的尚未鉴定的组分（再活化因子）重新活化。pI为5.80，测定了ATP、Mg ~（2+）和DL-硫辛酸的Km值。硒代脂肪酸和6-硫代辛酸是替代的，但较差的底物。脂酰化被6-和8-硒代辛酸酯和8-硫代辛酸酯可逆地抑制，其与LPL的六个半胱氨酸巯基反应。LPL失活的Cu 2+离子在一个过程中，涉及形成的分子间和分子内的二硫键。对缺乏LPL活性的lplA突变体的研究表明，E.大肠杆菌具有另一个独特的脂酰化系统，尽管在体外没有检测到这种活性。
Lipoate is an essential component of the 2-oxoacid dehydrogenase complexes and the glycine-cleavage system of Escherichia coli. It is attached to specific lysine residues in the lipoyl domains of the E2p (lipoate acetyltransferase) subunit of the pyruvate dehydrogenase complex by a Mg2+- and ATP-dependent lipoate protein ligase (LPL). LPL was purified from wild-type E. coli, where its abundance is extremely low (< 10 molecules per cell) and from a genetically amplified source. The purified enzyme is a monomeric protein (M(r) 38 000) which forms irregular clusters of needle-like crystals. It is stable at -20 degrees C, but slowly oxidizes to an inactive form containing at least one intramolecular disulphide bond at 4 degrees C. The inactive form could be re-activated by reducing agents or by an as-yet unidentified component (reactivation factor) which is resolved from LPL at the final stage of purification. The pI is 5.80, and the K-m values for ATP, Mg2+ and DL-lipoate were determined. Selenolipoate and 6-thio-octanoate were alternative but poorer substrates. Lipoylation was reversibly inhibited by the 6- and 8-seleno-octanoates and 8-thio-octanoate, which reacted with the six cysteine thiol groups of LPL. LPL was inactivated by Cu2+ ions in a process that involved the formation of inter- and intra-molecular disulphide bonds. Studies with lplA mutants lacking LPL activity indicated that E. coli possesses another distinct lipoylation system, although no such activity could be detected in vitro.