el-Sayed M Y, DeBose C D, Coury L A, Roberts M F
Biochim Biophys Acta. 1985 Dec 4;837(3):325-35. doi: 10.1016/0005-2760(85)90056-6.
The structural features of a phosphatidylcholine molecule important for binding to phospholipase C (Bacillus cereus) have been examined using kinetic analyses of a series of short-chain phosphatidylcholines and analogues. Lipids examined had varying chain lengths, methyl branched chains, phenyl alkanoate chains, and a single fatty acyl chain (lysophosphatidylcholines). A comparison of Vmax and Km for monomolecularly dispersed dibutyroyl-, dihexanoyl- and diheptanoylphosphatidylcholine indicates that the length of the fatty acyl chains must be at least six carbons for efficient binding of the phosphatidylcholine to the enzyme. Enzymatic rates of hydrolysis for pure short-chain phosphatidylcholine micelles of different chain lengths or detergent mixed micelles with comparable concentrations of short- and long-chain phosphatidylcholines show no dependence on substrate chain length greater than six carbons. Methyl branching of short-chain phosphatidylcholines only inhibits phospholipase C activity when the methyl group is adjacent to the carbonyl (e.g., di(2-methyl)hexanoylphosphatidylcholine). In a similar fashion, phosphatidylcholines with phenylalkanoate chains become poor substrates when the phenyl group is near the acyl linkage. As the phenyl group is moved from C-4 to C-2 a large increase in the micellar apparent Km is observed. Chain specificity (sn-1 and/or sn-2 ester linkages) for binding is not absolute, since phospholipase C will hydrolyze micellar short-chain lysophosphatidylcholines at rates one tenth of phosphatidylcholines. In contrast, substitution of ester linkages with ether moieties yields phosphatidylcholine analogues which are even poorer substrates and not good inhibitors of phospholipase C. These results suggest that the carbonyl group and its immediate environment are important for phospholipid interacting with this water-soluble lipolytic enzyme.
通过对一系列短链磷脂酰胆碱及其类似物进行动力学分析，研究了磷脂酰胆碱分子与磷脂酶C（蜡样芽孢杆菌）结合的重要结构特征。所研究的脂质具有不同的链长、甲基支链、苯烷酸链以及单条脂肪酰链（溶血磷脂酰胆碱）。对单分子分散的二丁酰、二己酰和二庚酰磷脂酰胆碱的Vmax和Km进行比较表明，脂肪酰链的长度必须至少为六个碳，磷脂酰胆碱才能有效地与该酶结合。不同链长的纯短链磷脂酰胆碱胶束或具有相当浓度的短链和长链磷脂酰胆碱的去污剂混合胶束的酶促水解速率表明，底物链长大于六个碳时，水解速率与链长无关。短链磷脂酰胆碱的甲基支链只有在甲基与羰基相邻时（例如，二（2-甲基）己酰磷脂酰胆碱）才会抑制磷脂酶C的活性。同样，当苯基靠近酰基连接时，带有苯烷酸链的磷脂酰胆碱会成为不良底物。随着苯基从C-4移至C-2，胶束表观Km会大幅增加。结合的链特异性（sn-1和/或sn-2酯键）并非绝对，因为磷脂酶C水解胶束短链溶血磷脂酰胆碱的速率仅为磷脂酰胆碱的十分之一。相比之下，用醚部分取代酯键会产生磷脂酰胆碱类似物，这些类似物是更差的底物，也不是磷脂酶C的良好抑制剂。这些结果表明，羰基及其紧邻环境对于磷脂与这种水溶性脂解酶的相互作用很重要。
