Plückthun A, Dennis E A
Biochemistry. 1982 Apr 13;21(8):1750-6. doi: 10.1021/bi00537a008.
Phospholipase A2 from cobra venom (Naja naja naja), which acts poorly on phosphatidylethanolamine (PE) in mixed micelles, is activated toward PE by the monomeric phospholipid dibutyrylphosphatidylcholine (dibutyryl-PC) which is an, even poorer substrate. Phosphorus-31 nuclear magnetic resonance spectroscopy was employed to show that only PE is hydrolyzed in mixtures of PE and dibutyryl-PC of various concentrations. The activation shows saturation behavior, and the fully activated enzyme hydrolyzes PE at a rate similar to its optimal substrate PC containing long chain fatty acid groups. Because dibutyryl-PC is not incorporated into the micelles, these results are consistent with a mechanism of direct activation of the enzymes by dibutyryl-PC rather than a change in the properties of the interface being responsible for the activation of phospholipase A2. Furthermore, if either PC pr PE as substrate is dispersed in mixed micelles, increasing amounts of the detergent Triton X-100 decrease the hydrolysis rate. The same detergent effect occurs if PE hydrolysis is activated by sphingomyelin (SPH). However, if the enzyme is activated by the monomeric dibutyryl-PC, this detergent effect can be overcome at high enough dibutyryl-PC concentrations. The hydrolysis of the monomeric dibutyryl-PC can also be stimulated by SPH in mixed micelles. This reaction shows no effect of detergent. Several models are considered to explain these observations, and it is suggested that the enzyme has two types of functional sites: an activator site and a catalytic site.
眼镜蛇毒（眼镜蛇指名亚种）中的磷脂酶A2对混合胶束中的磷脂酰乙醇胺（PE）作用较弱，但可被单体磷脂二丁酰磷脂酰胆碱（二丁酰磷脂酰胆碱，一种更差的底物）激活。采用磷-31核磁共振波谱法表明，在不同浓度的PE和二丁酰磷脂酰胆碱混合物中，只有PE被水解。这种激活表现出饱和行为，完全激活的酶水解PE的速率与其最佳底物含长链脂肪酸基团的磷脂酰胆碱相似。由于二丁酰磷脂酰胆碱不掺入胶束中，这些结果与二丁酰磷脂酰胆碱直接激活酶的机制一致，而不是界面性质的改变导致磷脂酶A2的激活。此外，如果以磷脂酰胆碱或PE作为底物分散在混合胶束中，增加去污剂Triton X-100的量会降低水解速率。如果鞘磷脂（SPH）激活PE水解，也会出现同样的去污剂效应。然而，如果酶被单体二丁酰磷脂酰胆碱激活，在足够高的二丁酰磷脂酰胆碱浓度下可以克服这种去污剂效应。混合胶束中二丁酰磷脂酰胆碱单体的水解也可被鞘磷脂刺激。该反应不受去污剂影响。考虑了几种模型来解释这些观察结果，并且认为该酶有两种功能位点：一个激活位点和一个催化位点。
