AN UNUSUAL ACTIVE-SITE IDENTIFIED IN A FAMILY OF ZINC METALLOENDOPEPTIDASES
AN UNUSUAL ACTIVE-SITE IDENTIFIED IN A FAMILY OF ZINC METALLOENDOPEPTIDASES
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DOI：
10.1073/pnas.89.9.3835
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发表时间：
1992-05-01
影响因子：
11.1
通讯作者：
ROTH, RA
中科院分区：
文献类型：
--
作者：
BECKER, AB;ROTH, RA
在锌金属内肽酶家族中发现了一个不寻常的活性位点，包括细菌蛋白酶III和人类和果蝇的胰岛素降解酶。所有这些酶都被定性为金属内肽酶，纯化的蛋白酶III已被证明含有化学计量水平的锌。然而，这三种蛋白酶都缺乏其他锌金属内肽酶活性位点所描述的一致序列（HEXXH）。相反，这些蛋白酶含有该基序的反转，HXXEH。为了确定该区域是否代表这些蛋白的活性位点，我们分别将蛋白酶III中的两种组氨酸突变为精氨酸，谷氨酸突变为谷氨酰胺。与野生型蛋白酶相比，这三种突变体都缺乏对外源底物胰岛素的蛋白水解活性。有三条证据表明，突变体中活性的丧失并非由于蛋白质三维结构的扭曲：(i)突变体正常分泌到质周空间和色谱仪中；（ii）所有三种突变体的表达水平几乎与野生型蛋白质相同，并且似乎对细菌的蛋白质水解没有增加的敏感性；（iii）在放射免疫分析中，突变体与野生型蛋白平等竞争。通过原子吸收分光光度法发现纯化的野生型和谷氨酸突变体含有化学计量量的锌，而组氨酸突变体的锌信号可以忽略不计。这些发现与该区域是该蛋白的活性位点一致，组氨酸残基协调必需的锌原子和谷氨酸参与催化。
An unusual active site has been identified in a family of zinc metalloendopeptidases that includes bacterial protease III and the human and Drosophila insulin-degrading enzymes. All of these enzymes have been characterized as metalloendopeptidases and purified protease III has been shown to contain stoichiometric levels of zinc. However, all three proteases lack the consensus sequence (HEXXH) described in the active site of other zinc metalloendopeptidases. Instead, these proteases contain an inversion of this motif, HXXEH. To determine whether this region could represent the active site in these proteins, the two histidines in protease III were individually mutated to arginine and the glutamate was mutated to glutamine. All three mutants were devoid of proteolytic activity toward an exogenous substrate, insulin, as compared to the wild-type protease. Three lines of evidence indicate that this loss of activity in the mutants is not due to distortion of the three-dimensional structure of the protein: (i) the mutants are secreted into the periplasmic space and chromatograph normally; (ii) all three mutants are expressed at levels nearly identical to wild-type protein and do not appear to have an increased susceptibility to proteolysis in the bacteria; and (iii) the mutants compete equally with wild-type protein in a radioimmunoassay. The purified wild-type and glutamate mutants were found to contain stoichiometric amounts of zinc by atomic absorption spectrophotometry, whereas both histidine mutants had negligible zinc signals. These findings are consistent with this region being the active site in this protein, with the histidine residues coordinating the essential zinc atom and the glutamate involved in catalysis.