Brahatheeswaran B, Prakash V, Savithri H S, Rao N A
Department of Biochemistry, Indian Institute of Science, Bangalore, India.
Arch Biochem Biophys. 1996 Jun 15;330(2):363-72. doi: 10.1006/abbi.1996.0263.
Sheep liver serine hydroxymethyltransferase (EC 2.1.2.1) is a homotetramer of Mr 213,000 requiring pyridoxal-5'-phosphate (PLP) as cofactor. Removal of PLP from the holoenzyme converted the enzyme to the apo form which, in addition to being inactive, was devoid of the characteristic absorption spectrum. Upon the addition of PLP to the apoenzyme, complete activity was restored and the visible absorption spectrum with a maximum at 425 nm was regained. The interaction of PLP with the apoenzyme revealed two phases of reaction with pseudo-first-order rate constants of 20 +/- 5 s(-1) and 12.2 +/- 2.0 x 10(-3) s(-1), respectively. However, addition of PLP to the apoenzyme did not cause gross conformational changes as evidenced by circular dichroic and fluorescence spectroscopy. Although conformationally apoenzyme and holoenzyme were indistinguishable, they had distinct apparent melting temperatures of 51 +/- 2 and 58 +/- 2 degrees C, respectively, and the reconstituted holoenzyme was thermally as stable as the native holoenzyme. These results suggested that there was no apparent difference in the secondary structure of holoenzyme, apoenzyme, and reconstituted holoenzyme. However, sedimentation analysis of the apoenzyme revealed the presence of two peaks of S20,w values of 8.7 +/- 0.5 and 5.7 +/- 0.3 S, respectively. A similar pattern was observed when the apoenzyme was chromatographed on a calibrated Sephadex G-150 column. The first peak corresponded to the tetrameric form (Mr 200,000 +/- 15,000) while the second peak had a Mr of 130,000 +/- 10,000. Reconstitution experiments revealed that only the tetrameric form of the apoenzyme could be converted into an active holoenzyme while the dimeric form could not be reconstituted into an active enzyme. These results demonstrate that PLP plays an important role in maintaining the structural integrity of the enzyme by preventing the dissociation of the enzyme into subunits, in addition to its function in catalysis.
绵羊肝脏丝氨酸羟甲基转移酶（EC 2.1.2.1）是一种分子量为213,000的同四聚体，需要磷酸吡哆醛（PLP）作为辅因子。从全酶中去除PLP会将酶转化为脱辅基形式，这种形式不仅无活性，而且没有特征吸收光谱。向脱辅基酶中添加PLP后，酶的活性完全恢复，并且重新获得了在425 nm处有最大值的可见吸收光谱。PLP与脱辅基酶的相互作用显示出两个反应阶段，其假一级速率常数分别为20±5 s⁻¹和12.2±2.0×10⁻³ s⁻¹。然而，如圆二色光谱和荧光光谱所示，向脱辅基酶中添加PLP不会引起明显的构象变化。尽管脱辅基酶和全酶在构象上难以区分，但它们的表观解链温度分别为51±2和58±2℃，且重组后的全酶与天然全酶一样热稳定。这些结果表明，全酶、脱辅基酶和重组全酶的二级结构没有明显差异。然而，对脱辅基酶的沉降分析显示存在两个峰，其S20,w值分别为8.7±0.5和5.7±0.3 S。当脱辅基酶在经校准的葡聚糖G - 150柱上进行色谱分析时，观察到类似的模式。第一个峰对应四聚体形式（分子量200,000±15,000），而第二个峰的分子量为130,000±10,000。重组实验表明，只有脱辅基酶的四聚体形式可以转化为有活性的全酶，而二聚体形式不能重组为有活性的酶。这些结果表明，PLP除了在催化中起作用外，还通过防止酶解离成亚基在维持酶的结构完整性方面发挥重要作用。
