The choline binding site of phospholipase C (Bacillus cereus): insights into substrate specificity.
The choline binding site of phospholipase C (Bacillus cereus): insights into substrate specificity.
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DOI：
10.1021/bi9919798
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发表时间：
2000-03
期刊：
影响因子：
2.9
通讯作者：
S. F. Martín;B. Follows;P. Hergenrother;B. Trotter
中科院分区：
文献类型：
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作者：
S. F. Martín;B. Follows;P. Hergenrother;B. Trotter
来自蜡样芽胞杆菌 (PLC(Bc)) 的磷脂酰胆碱偏好型磷脂酶 C 是一种 28.5 kDa 的酶，其活性位点具有三个锌离子。许多氨基酸残基作为锌配体以及在结合和催化中发挥的作用已被阐明。最近的机理研究表明，反应速率受到化学水解过程中质子转移步骤的限制，而不是底物结合或产物释放的限制。 PLC(Bc) 与磷脂酰胆碱相关磷酸酯抑制剂复合的 X 射线结构表明，三个氨基酸残基 Glu4、Tyr56 和 Phe66 构成胆碱结合袋。然而，由于这三个残基对底物识别和特异性的贡献尚不清楚，因此通过PCR诱变构建了一系列Glu4、Tyr56和Phe66的位点特异性突变体。根据各自 CD 光谱和解链温度的比较，突变体似乎在溶液中采用了与野生型 PLC(Bc) 几乎相同的折叠结构。三种可溶性底物 1, 2-二己酰基-sn-甘油-3-磷酸胆碱 (C6PC)、1, 2-二己酰基-sn-甘油-3-磷酸乙醇胺 (C6PE) 和 1, 2-二己酰基-sn-甘油-3-磷酸-L-丝氨酸水解的动力学参数 k(cat) 和 K(m)对于每个突变体，测定了低于其相应临界胶束浓度（cmc）值的浓度（C6PS）。用非芳香族残基替换 Phe66 会显着降低 k(cat)（大约 200 倍）并降低 PLC(Bc) 对 C6PC、C6PE 和 C6PS 的活性，而 Glu4 和 Tyr56 的改变通常会导致催化效率的损失更小。 Glu4 突变对 C6PS 的 k(cat) 和 K(m) 影响相对较小，但对 C6PC 和 C6PE 的 K(m) 影响显着。用非芳香族残基替换 Tyr56 也会影响催化效率，尽管程度比第 66 位的相应变化小得多。然而，第 56 位芳香族残基的存在似乎赋予 C6PC 和 C6PE 一些底物选择性，C6PC 和 C6PE 在头基上带有正电荷，而 C6PS 在头基上没有净电荷；这种特异性的增加主要是由于 C6PS 的 k(cat) 降低。
The phosphatidylcholine-preferring phospholipase C from Bacillus cereus (PLC(Bc)) is a 28.5 kDa enzyme with three zinc ions in its active site. The roles that a number of amino acid residues play as zinc ligands and in binding and catalysis have been elucidated. Recent mechanistic studies indicate that the rate of the reaction is limited by a proton-transfer step during chemical hydrolysis and not substrate binding or product release. An X-ray structure of PLC(Bc) complexed with a phosphonate inhibitor related to phosphatidylcholine revealed that the three amino acid residues Glu4, Tyr56, and Phe66 comprise the choline binding pocket. However, because the contributions that these three residues make to substrate recognition and specificity were unknown, a series of site-specific mutants for Glu4, Tyr56, and Phe66 were constructed by PCR mutagenesis. On the basis of a comparison of their respective CD spectra and melting temperatures, it appears that the mutants adopt folded structures in solution that are virtually identical to that of wild-type PLC(Bc). The kinetic parameters k(cat) and K(m) for the hydrolysis of the three soluble substrates 1, 2-dihexanoyl-sn-glycero-3-phosphocholine (C6PC), 1, 2-dihexanoyl-sn-glycero-3-phosphoethanolamine (C6PE), and 1, 2-dihexanoyl-sn-glycero-3-phospho-L-serine (C6PS) at concentrations below their corresponding critical micelle concentration (cmc) values were determined for each mutant. Replacement of Phe66 with a nonaromatic residue dramatically decreased k(cat) (approximately 200-fold) and reduced PLC(Bc) activity toward C6PC, C6PE, and C6PS, whereas changes to Glu4 and Tyr56 typically led to much more modest losses in catalytic efficiencies. Mutations of Glu4 had relatively little effect upon k(cat) and K(m) for C6PS, but they significantly influenced K(m) for C6PC and C6PE. Replacing Tyr56 with nonaromatic residues also affects catalytic efficiency, albeit to a much lesser degree than the corresponding changes at position 66. However, the presence of an aromatic residue at position 56 seems to confer some substrate selectivity for C6PC and C6PE, which bear a positive charge on the headgroup, relative to C6PS, which has no net charge on the headgroup; this increase in specificity arises largely from a reduced k(cat) for C6PS.