权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Enzymatic and protein studyes of penicillolysin, a new 18 k metalloendopeptidase from Penicillium citrinum

青霉溶血素（一种来自柑橘青霉的新型 18 k 金属内肽酶）的酶学和蛋白质研究

基本信息

批准号：
06660086
负责人：
ICHISHIMA Eiji
金额：
$ 1.34万
依托单位：
TOHOKU UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1994
资助国家：
日本
起止时间：
1994 至 1995
项目状态：
已结题

项目摘要

The zinc atom is an essential component of penicillolysin. Earlier studies found that the enzyme contains 1g-atom of zinc per mole of enzyme (Mr 17,000). The alpha-helix and beta-structure contents of the enzyme are ca 19 and 58%, respectively. The enzyme showed high affinity towards the Pro-X(X=Gln, Lys, Leu or Arg) bonds of substance P,dynorphine A,neurotensin and chicken brain pentapeptide, and the Arg-Arg bonds in dynorphine A and neuroteinsin. Preferential cleavages of bonds by the enzyme with hydrophobic amino acid residues at the P_1 position are observed on the peptide used. The specificity of penicillolysin differs from those of other metalloendopeptidase.Site-directed mutagenesis of penicillolysin cDNAwas used to assess the functional role of catalytic residues. Our results demonstrate that a single amino acid substitution of Asn for Asp-121 or Asp-164 resulting in mutant enzymes causes drastic decrease in the catalysis of self-maturation with Zn^<2+> ions. The two mutants had no activity of clear zone formation. These results strongly support that Asp-121 and Asp-164 in penicillolysin are crucial for hydrolytic activity. The two essential aspartic acid residues differ from those of the thermolysin family. To identify the zinc ligands, we substituted Ala for His-128 or His-132. The result shows that His-128 and His-132 are essential amino acid residues and are identical to those of the zinc binding motif, HEXXH of the thermolysin family. Our result also shows that Glu-129 is an essential amino acid residue, while Glu-65 is not.It is concluded that Asp-121, Asp-164 and Glu-129 are crucial for the hydrolytic activity of penicillolysin, that His-128 and His-132 are also crucial residues for zinc-coordinating enzymes, and that Asp-104 and Asp-143 may be binding sites of the enzyme towards basic substrate.

锌原子是青霉素溶素的重要组成部分。早期的研究发现，这种酶每摩尔含有1克锌原子（Mr 17，000）。该酶的α-螺旋和β-结构含量分别约为19%和58%。该酶对P物质、强啡肽A、神经降压素和鸡脑五肽的Pro-X（X=Gln、Lys、Leu或Arg）键，以及强啡肽A和神经降压素的Arg-Arg键都有很高的亲和力。在所用的肽上观察到在P_1位具有疏水氨基酸残基的酶对键的优先裂解。青霉素溶素的特异性不同于其他金属内肽酶，因此，利用定点突变技术对青霉素溶素cDNA进行突变，以确定其催化残基的功能。我们的研究结果表明，Asn取代Asp-121或Asp-164导致突变酶的单个氨基酸会导致Zn^2+离子自成熟催化作用的急剧下降。这两个突变体没有形成透明带的活性。这些结果有力地支持了青霉素溶素中的Asp-121和Asp-164对于水解活性是至关重要的。两个必需的天冬氨酸残基不同于嗜热菌蛋白酶家族的那些。为了鉴定锌配体，我们用Ala取代His-128或His-132。结果表明，His-128和His-132是必需氨基酸残基，并且与嗜热菌蛋白酶家族的锌结合基序HEXXH的氨基酸残基相同。Glu-129是青霉素水解酶的必需氨基酸残基，而Glu-65不是必需氨基酸残基，因此推测Asp-121、Asp-164和Glu-129是青霉素水解酶活性的关键残基，His-128和His-132也是锌配位酶的关键残基，Asp-104和Asp-143可能是该酶与碱性底物的结合位点。