Histidine 282 in 5-aminolevulinate synthase affects substrate binding and catalysis.
Histidine 282 in 5-aminolevulinate synthase affects substrate binding and catalysis.
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5-氨基乙酰丙酸合酶中的组氨酸 282 影响底物结合和催化。
DOI：
10.1021/bi062053k
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发表时间：
2007
期刊：
影响因子：
2.9
通讯作者：
Ferreira,GloriaC
中科院分区：
文献类型：
--
作者：
Turbeville,TracyD;Zhang,Junshun;Hunter,GregoryA;Ferreira,GloriaC
5-氨基乙酰丙酸合酶（Aminolevulinate synthase，ALAS）是哺乳动物细胞中血红素生物合成途径的第一个酶，属于吡哆醛5 '-磷酸（pyridoxal 5'-phosphate，PLP）依赖性α-氧代胺合酶家族。在α-氧代胺合酶家族的所有酶的结构中，保守的组氨酸与PLP辅因子的酚氧形成氢键，并且对于底物结合、PLP定位和亚胺氮的pKa的维持可能是重要的。在ALAS中，用丙氨酸替换等同的组氨酸H282使甘氨酸的催化效率降低450倍，并使甘氨酸结合的慢相速率降低85%。随着A420/A330比值从0.45增加到1.05，吸收420和330 nm物质的分布发生改变。物种分布的这种变化反映在辅因子荧光和300 - 500 nm圆二色性光谱中，可能反映了全酶互变异构体分布的变化。ALAS和H282 A的300 - 500 nm圆二色光谱在甘氨酸或氨基乙酰丙酸存在下发散，表明PLP辅因子在外部醛亚胺形成时的重定向在H282 A中受阻。此外，还观察到 值和光谱和动力学性质，而 增加了9倍。总之，结果表明，H282协调的吡啶环的运动与重组的活性位点氢键网络，并作为一个氢键供体的酚氧，以保持质子化的席夫碱和增强的PLP辅因子的电子汇功能。
5-Aminolevulinate synthase (ALAS), the first enzyme of the heme biosynthetic pathway in mammalian cells, is a member of the α-oxoamine synthase family of pyridoxal 5‘-phosphate (PLP)-dependent enzymes. In all structures of the enzymes of the α-oxoamine synthase family, a conserved histidine hydrogen bonds with the phenolic oxygen of the PLP cofactor and may be significant for substrate binding, PLP positioning, and maintenance of the pKaof the imine nitrogen. In ALAS, replacing the equivalent histidine, H282, with alanine reduces the catalytic efficiency for glycine 450-fold and decreases the slow phase rate for glycine binding by 85%. The distribution of the absorbing 420 and 330 nm species was altered with anA420/A330ratio increased from 0.45 to 1.05. This shift in species distribution was mirrored in the cofactor fluorescence and 300−500 nm circular dichroic spectra and likely reflects variation in the tautomer distribution of the holoenzyme. The 300−500 nm circular dichroism spectra of ALAS and H282A diverged in the presence of either glycine or aminolevulinate, indicating that the reorientation of the PLP cofactor upon external aldimine formation is impeded in H282A. Alterations were also observed in the value and spectroscopic and kinetic properties, while the increased 9-fold. Altogether, the results imply that H282 coordinates the movement of the pyridine ring with the reorganization of the active site hydrogen bond network and acts as a hydrogen bond donor to the phenolic oxygen to maintain the protonated Schiff base and enhance the electron sink function of the PLP cofactor.