Molluscan chymotrypsin-like protease: structure, localization, and substrate specificity.
Molluscan chymotrypsin-like protease: structure, localization, and substrate specificity.
复制标题
软体动物胰凝乳蛋白酶样蛋白酶：结构、定位和底物特异性。
DOI：
10.1006/abbi.1993.1406
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发表时间：
1993
影响因子：
3.9
通讯作者：
Morse,DE
中科院分区：
文献类型：
--
作者：
Groppe,JC;Morse,DE
编码胰凝乳蛋白酶样前蛋白酶原的信使RNA在软体动物红鲍（Haliotis rufescens）肠的远端四分之一中大量且特异性地表达。与该发现一致，在该段肠腔中以最高浓度检测到胰凝乳蛋白酶样活性。由于在远端肠液中的总蛋白的复杂性是低的，糜蛋白酶样蛋白酶是高度表达的，纯化的酶，以接近同质性，实现了通过一个单一的通道上的阴离子交换树脂。通过水解具有不同P1氨基酸的四肽底物家族，证实了该蛋白酶的主要特异性，该蛋白酶的底物结合位点中的关键氨基酸残基（Ser 189，Gly 216和Ser 226）与其他胰凝乳蛋白酶样酶的S1亚位点中的类似残基具有同源性。最佳的P1残基包括具有与酶原的活化肽的P1处的天冬酰胺残基的侧链类似的大的γ-分支侧链（苯丙氨酸和亮氨酸）的那些残基。因此，与通过普通胰蛋白酶机制激活的胰腺丝氨酸蛋白酶的酶原不同，软体动物酶的酶原似乎通过自催化机制激活，即，通过用活性胰凝乳蛋白酶样蛋白酶切割。从一级序列预测的酶的其他独特性质包括未配对的半胱氨酸，具有蛋白酶的两种不同构象异构体之间的内部硫醇-二硫键异构化的潜力。
A messenger RNA encoding a chymotrypsin-like preproprotease is expressed abundantly and specifically in the distal quarter of the intestine of the molluseHaliotis rufescens(red abalone). Consistent with this finding, a chymotrypsin-like activity was detected at highest concentration in the lumen of this segment of the intestine. Because the complexity of total protein in the distal intestinal fluid was low and the chymotrypsin-like protease was highly expressed, purification of the enzyme to near homogeneity was achieved by a single passage over an anion-exchange resin. The primary specificity of the protease, predicted from homology of the key amino acid residues (Serl89, Gly216, and Ser226) in the substrate binding site with similar residues lining the S1subsites of other chymotrypsin-like enzymes, was confirmed by hydrolysis of a family of tetrapeptide substrates with different P1amino acids. The optimal P1residues include those with bulky, γ-branched side chains (phenylalanine and leucine) similar to the side chain of the asparagine residue at P1of the activation peptide of the proenzyme. Thus, unlike zymogens of the pancreatic serine proteases, which are activated by the common tryptic mechanism, the zymogen of the molluscan enzyme appears to be activated by an autocatalytic mechanism, i.e., by cleavage with active chymotrypsin-like protease. Additional unique properties of the enzyme predicted from the primary sequence include an unpaired cysteine, with the potential for internal thiol-disulfide isomerization between two different conformers of the protease.