Catalytically active alkaline molten globular enzyme: Effect of pH and temperature on the structural integrity of 5-aminolevulinate synthase.
Catalytically active alkaline molten globular enzyme: Effect of pH and temperature on the structural integrity of 5-aminolevulinate synthase.
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催化活性碱性熔球酶：pH 和温度对 5-氨基乙酰丙酸合酶结构完整性的影响。
DOI：
10.1016/j.bbapap.2014.09.013
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发表时间：
2014
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：
Stojanovski,BoskoM;Breydo,Leonid;Hunter,GregoryA;Uversky,VladimirN;Ferreira,GloriaC
5-氨基酮戊酸合成酶(ALAS)是一种依赖于吡哆醛-5‘磷酸(PLP)的酶，催化哺乳动物体内血红素生物合成的第一步。用圆二色谱(CD)和荧光光谱研究了pH(1.0-3.0和7.5-10.5)和温度(20和37℃)对AlAs结构完整性的影响。从远紫外CD推导出的二级结构对pH和温度变化大多有弹性。在pH=2.0时观察到部分去折叠，但进一步降低pH会导致酸诱导的二级结构重折叠到接近天然水平。用近紫外CD监测的三级结构刚性在酸性和特定的碱性条件下(pH=10.5和pH=9.5/37°C)失去，在那里AlAs以熔融的球状状态存在。随着碱度的增加，酶的结构变得不那么规整，内乙二胺的手性环境也发生了改变，从420 nm移动到330 nm的二色性带。在酸性条件下，PLP辅因子与ALAS解离。与8-苯胺基-1-萘磺酸的反应证实了碱性和酸性熔融球体中疏水团簇的增加，尽管后者的反应更为明显。此外，在pH=1.0和9.5时，用丙烯酰胺猝灭AlAs的本征荧光产生的动态猝灭常数略有不同。AlAs的碱性熔融球状状态是催化活性的(pH=9.5/37°C)，尽管Kcat值显著降低。最后，5-氨基乙酰丙酸的结合限制了碱性熔融球体中的构象波动。总体而言，我们的发现证明了ALAS的结构可塑性如何有助于获得一种功能酶。
5-Aminolevulinate synthase (ALAS), a pyridoxal-5′phosphate (PLP)-dependent enzyme, catalyzes the first step of heme biosynthesis in mammals. Circular dichroism (CD) and fluorescence spectroscopies were used to examine the effects of pH (1.0–3.0 and 7.5–10.5) and temperature (20 and 37 °C) on the structural integrity of ALAS. The secondary structure, as deduced from far-UV CD, is mostly resilient to pH and temperature changes. Partial unfolding was observed at pH 2.0, but further decreasing pH resulted in acid-induced refolding of the secondary structure to nearly native levels. The tertiary structure rigidity, monitored by near-UV CD, is lost under acidic and specific alkaline conditions (pH 10.5 and pH 9.5/37 °C), where ALAS populates a molten globule state. As the enzyme becomes less structured with increased alkalinity, the chiral environment of the internal aldimine is also modified, with a shift from a 420 nm to 330 nm dichroic band. Under acidic conditions, the PLP cofactor dissociates from ALAS. Reaction with 8-anilino-1-naphthalenesulfonic acid corroborates increased exposure of hydrophobic clusters in the alkaline and acidic molten globules, although the reaction is more pronounced with the latter. Furthermore, quenching the intrinsic fluorescence of ALAS with acrylamide at pH 1.0 and 9.5 yielded subtly different dynamic quenching constants. The alkaline molten globule state of ALAS is catalytically active (pH 9.5/37 °C), although thekcatvalue is significantly decreased. Finally, the binding of 5-aminolevulinate restricts conformational fluctuations in the alkaline molten globule. Overall, our findings prove how the structural plasticity of ALAS contributes to reaching a functional enzyme.