Chang L S, Kuo K W, Lin S R, Chang C C
Department of Biochemistry, Kaohsiung Medical College, Taiwan.
J Protein Chem. 1994 Oct;13(7):641-8. doi: 10.1007/BF01890463.
Phospholipase A2 (PLA2) from Bungarus multicinctus snake venom was subjected to Lys modification with 4-chloro-3,5-dinitrobenzoate and trinitrobenzene sulfonic acid, and one major carboxydinitrophenylated (CDNP) PLA2 and two trinitrophenylated (TNP) derivatives (TNP-1 and TNP-2) were separated by high-performance liquid chromatography. The results of amino acid analysis and sequence determination revealed that CDNP-PLA2 and TNP-1 contained one modified Lys residue at position 6, and both Lys-6 and Lys-62 were modified in TNP-2. It seemed that the Lys-6 was more accessible to modified reagents than other Lys residues in PLA2. Modification of Lys-6 caused a 94% drop in enzymatic activity as observed with CDNP-PLA2 and TNP-1. Alternatively, the enzyme modified on both Lys-6 and Lys-62 retained little PLA2 activity. Either carboxydinitrophenylation or trinitrophenylation did not significantly affect the secondary structure of the enzyme molecule as revealed by the CD spectra, and Ca2+ binding and antigenicity of Lys-6-modified PLA2 were unaffected. Conversion of nitro groups to amino groups resulted in a partial restoration of enzymatic activity of CDNP-PLA2 to 32% of that of PLA2. It reflected that the positively charged side chain of Lys-6 might play an exclusive role in PLA2 activity. The TNP derivatives could be regenerated with hydrazine hydrochloride. The biological activity of the regenerated PLA2 is almost the same as that of native PLA2. These results suggest that the intact Lys-6 is essential for the enzymatic activity of PLA2, and that incorporation of a bulky CDNP or TNP group on Lys-6 might give rise to the distortion of the interaction between substrate and the enzyme molecule, and the active conformation of PLA2.
用4-氯-3,5-二硝基苯甲酸和三硝基苯磺酸对多环眼镜蛇毒中的磷脂酶A2（PLA2）进行赖氨酸修饰，通过高效液相色谱法分离得到一种主要的羧基二硝基苯基化（CDNP）PLA2和两种三硝基苯基化（TNP）衍生物（TNP-1和TNP-2）。氨基酸分析和序列测定结果表明，CDNP-PLA2和TNP-1在第6位含有一个修饰的赖氨酸残基，而TNP-2中赖氨酸-6和赖氨酸-62均被修饰。似乎赖氨酸-6比PLA2中的其他赖氨酸残基更容易被修饰试剂作用。如CDNP-PLA2和TNP-1所示，赖氨酸-6的修饰导致酶活性下降94%。另外，在赖氨酸-6和赖氨酸-62上均被修饰的酶几乎没有PLA2活性。圆二色光谱显示，羧基二硝基苯基化或三硝基苯基化均未显著影响酶分子的二级结构，赖氨酸-6修饰的PLA2的钙离子结合能力和抗原性也未受影响。将硝基转化为氨基可使CDNP-PLA2的酶活性部分恢复至PLA2活性的32%。这表明赖氨酸-6带正电荷的侧链可能在PLA2活性中起独特作用。TNP衍生物可用盐酸肼再生。再生的PLA2的生物学活性与天然PLA2几乎相同。这些结果表明，完整的赖氨酸-6对PLA2的酶活性至关重要，在赖氨酸-6上引入庞大的CDNP或TNP基团可能会导致底物与酶分子之间相互作用以及PLA2活性构象的扭曲。
