Conversion of 5-aminolevulinate synthase into a more active enzyme by linking the two subunits: spectroscopic and kinetic properties.
Conversion of 5-aminolevulinate synthase into a more active enzyme by linking the two subunits: spectroscopic and kinetic properties.
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通过连接两个亚基将 5-氨基乙酰丙酸合酶转化为活性更高的酶：光谱和动力学特性。
DOI：
10.1110/ps.041258305
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发表时间：
2005
期刊：
影响因子：
--
通讯作者：
Ferreira,GloriaC
中科院分区：
文献类型：
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作者：
Zhang,Junshun;Cheltsov,AntonV;Ferreira,GloriaC
二聚体5 -氨基乙酰丙酸合成酶（ALAS）是一种依赖于吡哆醛5 ' -磷酸（PLP）的酶，它的两个活性位点位于亚基界面上，每个亚基都有必需氨基酸的贡献。将两个亚基连接成一个单一的多肽链二聚体（2XALAS）产生的酶的周转率大约是野生型ALAS的7倍。研究了2XALAS的光谱和动力学性质，以探索与野生型ALAS相比，辅酶结构和动力学机制的差异，从而获得更活跃的酶。ALAS和2XALAS的吸收光谱在410和330 nm处最大，在pH ~ 7.5处，2XALAS的A410/ a330比更大。330 nm吸收波段在385 nm处有较强的荧光，而在510 nm处没有，表明330 nm的吸收物质是取代醛胺而不是希夫碱的烯丙胺形式。这两种酶的385 nm发射强度随pH值的增加而增加，单个pkf为~ 8.5，因此410 nm和330 nm的吸收物种分别属于酮胺和取代醛胺。喹诺酮中间体eq2的生成和衰变的瞬态动力学分析表明，尽管在ALAS和2XALAS中它们的速率相似，但在2XALAS催化的反应中该中间体的积累更大。总的来说，这些结果表明酮胺是辅酶的活性形式，在2XALAS中形成的辅酶结构比pH ~ 7.5时形成的辅酶结构更突出。
The two active sites of dimeric 5‐aminolevulinate synthase (ALAS), a pyridoxal 5′‐phosphate (PLP)‐dependent enzyme, are located on the subunit interface with contribution of essential amino acids from each subunit. Linking the two subunits into a single polypeptide chain dimer (2XALAS) yielded an enzyme with an approximate sevenfold greater turnover number than that of wild‐type ALAS. Spectroscopic and kinetic properties of 2XALAS were investigated to explore the differences in the coenzyme structure and kinetic mechanism relative to those of wild‐type ALAS that confer a more active enzyme. The absorption spectra of both ALAS and 2XALAS had maxima at 410 and 330 nm, with a greater A410/A330ratio at pH ∼7.5 for 2XALAS. The 330 nm absorption band showed an intense fluorescence at 385 nm but not at 510 nm, indicating that the 330 nm absorption species is the substituted aldamine rather than the enolimine form of the Schiff base. The 385 nm emission intensity increased with increasing pH with a single pKof ∼8.5 for both enzymes, and thus the 410 and 330 nm absorption species were attributed to the ketoenamine and substituted aldamine, respectively. Transient kinetic analysis of the formation and decay of the quinonoid intermediate EQ2indicated that, although their rates were similar in ALAS and 2XALAS, accumulation of this intermediate was greater in the 2XALAS‐catalyzed reaction. Collectively, these results suggest that ketoenamine is the active form of the coenzyme and forms a more prominent coenzyme structure in 2XALAS than in ALAS at pH ∼7.5.