PEPTIDE SUBSTRATE-SPECIFICITY OF THE MEMBRANE-BOUND METALLOPROTEASE OF LEISHMANIA
PEPTIDE SUBSTRATE-SPECIFICITY OF THE MEMBRANE-BOUND METALLOPROTEASE OF LEISHMANIA
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DOI：
10.1021/bi00495a015
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发表时间：
1990-10-30
期刊：
影响因子：
2.9
通讯作者：
BORDIER, C
中科院分区：
文献类型：
--
作者：
BOUVIER, J;SCHNEIDER, P;BORDIER, C
利什曼原虫的前鞭毛体表面蛋白酶（PSP）是一种中性膜结合锌酶。该蛋白酶没有外肽酶活性，并且不会切割大量在 P1'' 位点具有显色和荧光离去基团的底物。通过使用已知氨基酸序列的天然和合成肽来研究酶的底物特异性。 11个切割位点的鉴定表明，当疏水性残基位于P1''位点且碱性氨基酸残基位于P2''和P3''位点时，该酶优先在氨基侧切割肽。此外，酪氨酸残基常见于 P1 位点。然而，水解并不限于这些残留物。这些结果使得能够合成模型肽H2N-L-I-A-Y-L-K-K-A-T-COOH，其在酪氨酸和亮氨酸残基之间被PSP以1.8倍的kcat/Km比率切割。 106 M-1 s-1。此外，发现与原序列的最后四个氨基酸和成熟PSP的前五个残基重叠的合成九肽在预期位点被蛋白酶切割以释放成熟酶。这一结果表明蛋白酶激活可能存在自催化机制。最后，异羟肟酸酯衍生的二肽Cbz-Tyr-Leu-NHOH显示出竞争性抑制PSP，KI为17μM。
The promastigote surface protease (PSP) of Leishmania is a neutral membrane-bound zinc enzyme. The protease has no exopeptidase activity and does not cleave a large selection of substrates with chromogenic and fluorogenic leaving groups at the P1'' site. The substrate specificity of the enzyme was studied by using natural and synthetic peptides of known amino acid sequence. The identification of 11 cleavage sites indicates that the enzyme preferentially cleaves peptides at the amino side when hydrophobic residues are in the P1'' site and basic amino acid residues in the P2'' and P3'' sites. In addition, tyrosine residues are commonly found at the P1 site. Hydrolysis is not, however, restricted to these residues. These results have allowed the synthesis of a model peptide, H2N-L-I-A-Y-L-K-K-A-T-COOH, which is cleaved by PSP between the tyrosine and leucine residues with a kcat/Km ratio of 1.8 .times. 106 M-1 s-1. Furthermore, a synthetic nonapeptide overlapping the last four amino acids of the prosequence and the first five residues of mature PSP was found to be cleaved by the protease at the expected site to release the mature enzyme. This result suggests a possible autocatalytic mechanism for the activation of the protease. Finally, the hydroxamate-derivatized dipeptide Cbz-Tyr-Leu-NHOH was shown to inhibit PSP competitively with a KI of 17 .mu.M.