罗非鱼脯氨酸内肽酶的分离及抑制剂对其的作用机制

Purification of a Prolyl Endopeptidase from the Skeletal Muscle of Tilapia(Oreochromis spp) and Its Inhibitory Mechanism

采用硫酸铵盐析及柱层析技术,从罗非鱼肌肉中分离纯化得到分子质量约为85 ku的脯氨酸内肽酶(prolyl endopeptidase,PEP)。通过肽质量指纹质谱分析,获得13个肽片段,含128个氨基酸残基,结果显示,与伯氏朴丽鱼(Haplochromis burtoni)的PEP完全一致。该酶特异分解荧光底物Suc-Gly-ProMCA和Suc-Gly-Pro-Leu-Gly-Pro-MCA,PEP特异性抑制剂SUAM-14746和丝氨酸蛋白酶抑制剂PMSF可以抑制该酶的活性。PEP催化Suc-Gly-Pro-MCA水解反应的活化能(Ea)为47.42 k J/mol。SUAM-14746对PEP表现为竞争性抑制作用,抑制常数(KI)为1.91μmol/L。金属离子Zn^(2+)和Cu^(2+)对PEP的抑制类型均为混合型抑制,其中对游离酶的抑制常数(KI)分别为1.80 mmol/L和0.07 mmol/L,对酶-底物络合物的抑制常数(KIS)分别为2.33 mmol/L和1.17 mmol/L。

A PEP was purified from the skeletal muscle of tilapia （ Oreochromis spp） by ammonium sulfate fractionation and a series of column chromatographies. PEP was in homogeneity with molecular weight of approximately 85 ku on SDS - PAGE. Peptide mass fingerprinting （PMF） of PEP obtained 13 fragments including 128 amino acid residues which was identical to a PEP from Haplochromis burtoni, suggesting this enzyme is a prolyl endopeptidase. The enzyme specifically hydrolyzed fluorescent substrates Suc - Gly - Pro - MCA and Suc - Gly - Pro - Leu - Gly - Pro - MCA. Using Suc - Gly - Pro - MCA as substrate, the effect of proteinase inhibitors on PEP was investigated. The results showed that SUAM - 14746, a specific inhibitor of prolyl endopeptidase could strongly inhibit its activity while other proteinase inhibitors did not show any effects. Activation energy （Ea） of the enzyme was 47.42 kJ/mol. SUAM - 14746 competitively inhibited its activity with KI of 1.91 μmol/L. Metal ions Zn2＋ and Cu 2＋ both revealed mixed inhibition to PEP, and their inhibition constants （ K1 ） were 1.80 mmol/L and 0.07 mmot/L, while enzyme-substrate complexes （ KIS ） were 2.33 mmol/L and 1.17 mmol/L, respectively.