Pseudomonas mevalonii 3-hydroxy-3-methylglutaryl-CoA lyase: characterization of the isolated recombinant protein and investigation of the enzyme's cation requirements.
Pseudomonas mevalonii 3-hydroxy-3-methylglutaryl-CoA lyase: characterization of the isolated recombinant protein and investigation of the enzyme's cation requirements.
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梅瓦隆假单胞菌 3-羟基-3-甲基戊二酰辅酶 A 裂解酶：分离重组蛋白的表征和酶阳离子需求的研究。
DOI：
10.1021/bi00160a037
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发表时间：
1992
期刊：
影响因子：
2.9
通讯作者：
Miziorko,HM
中科院分区：
文献类型：
--
作者：
Narasimhan,C;Miziorko,HM
摘要：甲羟戊酸假单胞菌3-羟基-3-甲基戊二酰-CoA裂解酶已在大肠杆菌中以活性形式表达并纯化至均一。粗提物中的酶活性比同源表达系统报道的高30倍。在Q-Sepharose快速流动阴离子交换层析之后，代表蛋白质的原核形式的第一均质制备物的酶表现出70单位/mg的比活性。纯化的酶在-80 ℃下储存于20%甘油中时是稳定的。重组细菌酶与禽肝裂解酶抗血清发生交叉反应，表明两种蛋白质之间存在一定的序列同源性。该酶对（S）-HMG-CoA的Km= 20 pM。二价阳离子（Mg 2+和Mn 2+）显着刺激的酶活性测定条件下，活性只有适度增加外源硫醇。激活剂常数，Kn，Mg 2+（6.9 mM）是3个数量级大于Mn 2+（2.0 μ M）。虽然EDTA不影响活性，邻菲咯啉处理显着抑制酶。相比之下，邻菲咯啉是无效的，这表明邻位异构体的效果是由于螯合的紧密结合的金属离子。分离的酶的原子吸收和EPR分析表明存在紧密结合的铜。在使用标准LB肉汤表达的酶中，检测到化学计量仅为0.07-0.10的铜。当生长培养基补充有ImM CuS 〇 4时，铜结合的化学计量增加至每个酶亚基超过0.7。与金属含量低的酶相比，富铜裂解酶显示增强的热稳定性。原子吸收和EPR化学计量估计之间的协议表明，所有的紧密结合的阳离子是二价铜。EPR参数A = 152 G，与II型铜的归属一致。测得的g|值2.28表明阳离子结合涉及氮配体。3-羟基-3-甲基戊二酰-CoA裂解酶（EC 4.1. 3.4)催化真核肝生酮中的终末反应（Lynen等，1958），即3-羟基-3-甲基戊二酰-CoA（HMG-CoA）转化为乙酰乙酸和乙酰-CoA。因此，该酶在酮体的产生中起着至关重要的作用，酮体是饥饿期间燃料的替代来源（罗宾逊和威廉姆斯，
Revised Manuscript Received August 19, 1992 abstract: Pseudomonas mevalonii3-hydroxy-3-methylglutaryl-CoA lyase has beenexpressed in an active form in Escherichia coli and purified to homogeneity. Enzyme activity in crude extracts is 30-fold higher than reported for a homologous expression system. After Q-Sepharose fast-flow anion-exchange chromatography, the enzyme, which represents the first homogeneous preparation of a prokaryotic form of the protein, exhibits a specific activity of 70 units/mg. The purified enzyme is stable when stored in 20% glycerol at-80 C. The recombinant bacterial enzyme cross reacts with antiserum produced against avian liver lyase, indicating some sequence homology between the two proteins. The enzyme exhibits a Km= 20 pM for (S)-HMG-CoA. Divalent cations (Mg2+ and Mn2+) markedly stimulate theenzyme activity under assay conditions; activity is only modestly increased by exogenous mercaptans. The activator constant, Kn, for Mg2+(6.9 mM) is 3 orders of magnitude greater than that for Mn2+(2.0 jiM). While EDTA dos not affect activity, o-phenanthroline treatment markedly inhibits the enzyme. In contrast, w-phenanthroline is ineffective, suggesting that the ortho isomer’s effect is attributable to chelation of a tightly bound metal ion. Atomic absorption and EPR analyses of isolated enzyme indicate the presence of tightly bound copper. In enzyme expressed using standard LB broth, copper is detected at stoichiometries of only 0.07-0.10. When the growth medium is supplemented with 1 mM CuSC> 4, stoichiometry of copper binding increases to over 0.7 per enzyme subunit. Copper-enriched lyase displays enhanced thermal stability in comparison with enzyme that is low in metal content. Agreement between atomic absorptionand EPR stoichiometry estimates suggests that all of the tighly bound cation is divalent copper. The EPR parameter A\\is estimated to be152 G, compatible with assignment of type II copper. The measured g| value of 2.28 suggests that cation binding involves nitrogen ligand (s).3-Hydroxy-3-methylglutaryl-CoA lyase (EC 4.1. 3.4) cat-alyzes the terminal reaction in eukaryotic hepatic ketogenesis (Lynen et al., 1958), namely, the conversion of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) into acetoacetate and acetyl-CoA. The enzyme thus plays a vital role in the generation of ketone bodies which are alternative sources of fuel during periods of starvation (Robinson and Williams,
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DOI：
--
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