Purification and characterisation of an alpha-glucan phosphorylase from the thermophilic bacterium Thermus thermophilus.
Purification and characterisation of an alpha-glucan phosphorylase from the thermophilic bacterium Thermus thermophilus.
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DOI：
10.1111/j.1432-1033.1996.0150u.x
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发表时间：
1996-07
期刊：
影响因子：
--
通讯作者：
B. Boeck;R. Schinzel
中科院分区：
文献类型：
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作者：
B. Boeck;R. Schinzel
一种α-葡聚糖磷酸化酶已经从嗜热菌Thermus thermophilus中纯化了4500倍。与其他细菌磷酸化酶相比，嗜热酶似乎既不能被麦芽糖诱导，也不能被葡萄糖抑制。T.嗜热菌磷酸化酶与已知的嗜中温磷酸化酶共享主要特性，例如吡哆醛5 ′-磷酸含量（1 M吡哆醛-P/M亚基）、亚基分子量（约90 kDa）和抑制剂常数。T.在70 ° C下在pH 5.5-6.5范围内观察到嗜热菌磷酸化酶。虽然在25摄氏度的亚基组成的嗜热酶是一个八聚体的形式，在70摄氏度的最佳温度的优先形式似乎是一个二聚体。最值得注意的是，在合成和降解方向上，寡糖底物的极限尺寸比其他α-葡聚糖磷酸化酶降解的底物的最小尺寸短一个葡萄糖残基。麦芽四糖和糖原的降解速率与麦芽七糖相似（Vmax = 18 U/mg）。相应地，麦芽三糖在合成方向上充当引物。辅因子的荧光光谱和吸收光谱的差异以及砷酸根作为底物的失效表明T。嗜热菌磷酸化酶不同于已知的α-葡聚糖磷酸化酶。
An alpha-glucan phosphorylase has been purified 4500-fold from the thermophilic bacteria Thermus thermophilus. In contrast to other bacterial phosphorylases the thermophilic enzyme seems neither to be inducible by maltose nor repressed by glucose. T. thermophilus phosphorylase shares major properties with known mesophilic phosphorylases such as pyridoxal 5'-phosphate content (1 M pyridoxal-P/M subunit), subunit molecular mass (about 90 kDa) and inhibitor constants. The optimum temperature of T. thermophilus phosphorylase was observed at 70 degrees C in the pH range 5.5-6.5. While at 25 degrees C the subunit composition of the thermophilic enzyme is an octameric form, the preferential form at the optimum temperature of 70 degrees C seems to be a dimer. Most remarkably, in the direction of synthesis and degradation the limiting size of the oligosaccharide substrate is shorter by one glucose residue than the minimum size of substrate degraded by other alpha-glucan phosphorylases. Maltotetraose and glycogen are degraded with rates similar to that observed with maltoheptaose (Vmax = 18 U/mg). Correspondingly, maltotriose functions as primer in the synthesis direction. Differences in fluorescence and absorption spectra of the cofactor and the failure of arsenate acting as a substrate indicate that the active site structure of T. thermophilus phosphorylase differs from that of known alpha-glucan phosphorylases.