Barrett A J, Brown M A
Department of Biochemistry, Strangeways Research Laboratory, Cambridge, U.K.
Biochem J. 1990 Nov 1;271(3):701-6. doi: 10.1042/bj2710701.
Pz-peptidase was purified from chicken liver as a protein of Mr 80,000 and pI 5.2. The purified enzyme hydrolysed phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-D-Arg, 2,4-dinitrophenyl-Pro-Leu-Gly-Pro-Trp-D-Lys. 7-methoxycoumarin-3-carboxylyl-Pro-Leu-Gly-Pro-D-(2,4-dinitropheny l)Lys, benzoyl-Gly-Ala-Ala-Phe-p-aminobenzoate, Ac-Ala4 (at the Ala-1-Ala-2 bond) and bradykinin (at the Phe-5-Ser-6 bond). No hydrolysis of proteins was detected. Loss of activity in the presence of EDTA or 1,10-phenanthroline was time-dependent. Metal ions found to restore activity after treatment with EDTA were Zn2+, Mn2+, Ca2+, Co2+ and Cd2+, in decreasing order of effectiveness. Ni2+, Fe2+ and higher concentrations of Zn2+ were inhibitory. Inhibition by N-[1-(RS)-carboxy-3-phenylpropyl]-Ala-Ala-Tyr-p-aminobenzoate and related compounds showed Ki values (down to 5 nM) somewhat lower than those for the rat enzyme. Pz-peptidase was activated by low concentrations of 2-mercaptoethanol and dithiothreitol, but inhibited by higher concentrations. p-Chloromercuribenzoate and some other thiol-blocking reagents were inhibitory. Inactivation by diethyl pyrocarbonate that was reversible by hydroxylamine showed the presence of essential histidine residue(s). We conclude that chicken Pz-peptidase is a metallo-endopeptidase with thiol-dependence. Moreover, the properties of chicken Pz-peptidase agree with those described for mammalian soluble metallo-endopeptidase and endo-oligopeptidase A. consistent with the view that these three types of activity are all attributable to the single enzyme for which the name thimet peptidase has been proposed.
Pz-肽酶是从鸡肝中纯化得到的一种蛋白质，其相对分子质量为80,000，等电点为5.2。纯化后的酶可水解苯偶氮苄氧羰基-脯氨酸-亮氨酸-甘氨酸-脯氨酸-D-精氨酸、2,4-二硝基苯基-脯氨酸-亮氨酸-甘氨酸-脯氨酸-色氨酸-D-赖氨酸、7-甲氧基香豆素-3-羧基-脯氨酸-亮氨酸-甘氨酸-脯氨酸-D-(2,4-二硝基苯基)赖氨酸、苯甲酰-甘氨酸-丙氨酸-丙氨酸-苯丙氨酸-对氨基苯甲酸、Ac-Ala4（在丙氨酸-1-丙氨酸-2键处）和缓激肽（在苯丙氨酸-5-丝氨酸-6键处）。未检测到对蛋白质的水解作用。在EDTA或1,10-菲啰啉存在下，酶活性的丧失呈时间依赖性。经EDTA处理后发现能恢复活性的金属离子有Zn2+、Mn2+、Ca2+、Co2+和Cd2+，其有效性依次降低。Ni2+、Fe2+和较高浓度的Zn2+具有抑制作用。N-[1-(RS)-羧基-3-苯基丙基]-丙氨酸-丙氨酸-酪氨酸-对氨基苯甲酸及相关化合物的抑制作用显示其抑制常数（低至5 nM）略低于大鼠酶的抑制常数。低浓度的2-巯基乙醇和二硫苏糖醇可激活Pz-肽酶，但高浓度则具有抑制作用。对氯汞苯甲酸和其他一些巯基阻断试剂具有抑制作用。焦碳酸二乙酯的失活作用可被羟胺逆转，表明存在必需的组氨酸残基。我们得出结论，鸡Pz-肽酶是一种具有硫醇依赖性的金属内肽酶。此外，鸡Pz-肽酶的性质与哺乳动物可溶性金属内肽酶和内寡肽酶A的性质相符，这与这三种类型的活性均归因于已被提议命名为硫醚肽酶的单一酶的观点一致。
