Cengiz D, Cokuğraş A N, Kilinç K, Tezcan E F
Department of Biochemistry, Faculty of Medicine, Hacettepe University, Anhara, Turkey.
Biochem Mol Med. 1997 Jun;61(1):52-7. doi: 10.1006/bmme.1997.2578.
The effects of histidine-modifying reagents on human serum butyrylcholinesterase (BChE) were investigated. The commercially available enzyme was further purified by chromatography on a Sepharose CI-6B column prior to use. In the modification studies, we found that the histidine-specific reagents tosylphenylalanine chloromethyl ketone (TPCK) and tosyllysine chloromethyl ketone (TLCE) did not modify the enzyme; however, they inhibited the enzyme reversibly. The kinetic parameters of enzyme inhibition calculated were alpha = 10.8, beta = 0.26, and Ki = 0.016 mM for TPCK. TLCK inhibition gave similar kinetic behavior, with alpha = 41.6, beta = 0.065, and Ki = 0.039 mM. Tosyllysine, an analog of TLCK, did not inhibit the enzyme. Removal of TPCK and TLCK by dialysis resulted in significant reactivation of the enzyme. From kinetic studies, it was found that the inhibitions were hyperbolic mixed-type inhibitions. We concluded that the reagents competed with substrate for hydrophobic binding sites and inhibited the enzyme reversibly. On the other hand, in the modification studies with diethyl pyrocarbonate (DPC), it was observed that inactivation of the enzyme was irreversible and time-dependent. In the protection studies, the activity of the enzyme was partially protected from inactivation by DPC even at a 50 mM concentration of butyrylthiocholine. The results indicate that DPC modifies some essential histidine side chains in BChE, including the functional histidyl residue found at the active site.
研究了组氨酸修饰试剂对人血清丁酰胆碱酯酶（BChE）的影响。市售酶在使用前通过Sepharose CI - 6B柱色谱进一步纯化。在修饰研究中，我们发现组氨酸特异性试剂甲苯磺酰苯丙氨酸氯甲基酮（TPCK）和甲苯磺酰赖氨酸氯甲基酮（TLCK）不会修饰该酶；然而，它们可逆地抑制该酶。计算得到的TPCK酶抑制动力学参数为α = 10.8，β = 0.26，Ki = 0.016 mM。TLCK抑制表现出类似的动力学行为，α = 41.6，β = 0.065，Ki = 0.039 mM。TLCK的类似物甲苯磺酰赖氨酸不抑制该酶。通过透析去除TPCK和TLCK导致酶显著重新激活。从动力学研究发现，这些抑制是双曲线混合型抑制。我们得出结论，这些试剂与底物竞争疏水结合位点并可逆地抑制该酶。另一方面，在用焦碳酸二乙酯（DPC）进行的修饰研究中，观察到酶的失活是不可逆的且与时间相关。在保护研究中，即使在丁酰硫代胆碱浓度为50 mM时，该酶的活性也部分受到DPC失活的保护。结果表明DPC修饰了BChE中一些必需的组氨酸侧链，包括在活性位点发现的功能性组氨酸残基。
