Substrate and inhibitor studies of thermolysin-like neutral metalloendopeptidase from kidney membrane fractions. Comparison with bacterial thermolysin.
Substrate and inhibitor studies of thermolysin-like neutral metalloendopeptidase from kidney membrane fractions. Comparison with bacterial thermolysin.
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DOI：
10.1021/bi00354a015
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发表时间：
1986-03
期刊：
影响因子：
2.9
通讯作者：
M. Pozsgay;C. Michaud;M. Liebman;M. Orłowski
中科院分区：
文献类型：
--
作者：
M. Pozsgay;C. Michaud;M. Liebman;M. Orłowski
测定了一系列N-羧甲基肽抑制剂对兔肾膜结合金属内肽酶的抑制常数和一系列模型底物的动力学参数（Km、kcat和kcat/Km），并与细菌嗜热菌蛋白酶的抑制常数进行了比较。这两种酶显示出惊人的相似之处，在活性位点的S1'亚位点的疏水口袋结合底物的结构要求。这两种酶与在该位置具有亮氨酸残基的底物的反应速率最高，而苯丙氨酸残基的Km最低。这两种酶也被相同的N-羧甲基肽抑制剂抑制。尽管哺乳动物酶比其细菌对应物更容易受到抑制，但抑制剂分子的结构变异以类似的方式影响这两种酶的抑制常数。这两种酶显着不同，但是，相对于在P1和P2'位置的底物上的反应动力学参数的结构变化的效果。哺乳动物酶与P2、P1和P1 '位分别具有-Phe-Gly-Phe-或-Phe-Ala-Phe-的底物显示出最高的反应速率和特异性常数，而-Ala-Phe-Phe-是细菌酶最喜欢的序列。在脑啡肽中发现的序列--Gly-Gly-Phe--不受两种酶的影响。在P2'位具有氨基苯甲酸酯基的底物中，哺乳动物酶有利于在Meta位具有羧基的底物，而细菌酶有利于在帕拉具有羧基的底物。（250字处删节）
The inhibitory constants of a series of synthetic N-carboxymethyl peptide inhibitors and the kinetic parameters (Km, kcat, and kcat/Km) of a series of model synthetic substrates were determined for the membrane-bound kidney metalloendopeptidase isolated from rabbit kidney and compared with those of bacterial thermolysin. The two enzymes show striking similarities with respect to structural requirements for substrate binding to the hydrophobic pocket at the S1' subsite of the active site. Both enzymes showed the highest reaction rates with substrates having leucine residues in this position while phenylalanine residues gave the lowest Km. The two enzymes were also inhibited by the same N-carboxymethyl peptide inhibitors. Although the mammalian enzyme was more susceptible to inhibition than its bacterial counterpart, structural variations in the inhibitor molecules affected the inhibitory constants for both enzymes in a similar manner. The two enzymes differed significantly, however, with respect to the effect of structural changes in the P1 and P2' positions of the substrate on the kinetic parameters of the reaction. The mammalian enzyme showed the highest reaction rates and specificity constants with substrates having the sequence -Phe-Gly-Phe- or -Phe-Ala-Phe- in positions P2, P1, and P1', respectively, while the sequence -Ala-Phe-Phe- was the most favored by the bacterial enzyme. The sequence -Gly-Gly-Phe- as found in enkephalins was not favored by either of the enzymes. Of the substrates having an aminobenzoate group in the P2' position, the mammalian enzyme favored those with the carboxyl group in the meta position while the bacterial enzyme favored those with the carboxyl group in the para position.(ABSTRACT TRUNCATED AT 250 WORDS)