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

锌原子是青霉素溶菌素的重要组成部分。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、dynorphine A、神经紧张素和鸡脑五肽的Pro-X（X=Gln、Lys、Leu或Arg）键以及dynorphine A和神经紧张素的Arg-Arg键具有高亲和力。在所使用的肽上观察到具有P_1位疏水氨基酸残基的酶对键的优先切割。青霉溶酶的特异性不同于其他金属内肽酶。利用位点定向诱变技术对青霉溶酶cdna进行了研究，以评估催化残基的功能作用。我们的研究结果表明，Asn取代Asp-121或Asp-164的单个氨基酸导致突变酶导致Zn^<2+ b>离子对自成熟的催化作用急剧下降。这两个突变体没有形成清晰带的活性。这些结果有力地支持了青霉菌酵素中Asp-121和Asp-164对其水解活性至关重要。两种必需的天冬氨酸残基不同于热溶素家族的残基。为了鉴定锌配体，我们用Ala取代了His-128或His-132。结果表明，His-128和His-132是必需氨基酸残基，与热溶酶家族中锌结合基序HEXXH的氨基酸残基相同。我们的结果还表明，Glu-129是必需氨基酸残基，而Glu-65不是。结果表明，Asp-121、Asp-164和asp -129是青霉溶素水解活性的关键残基，His-128和His-132也是锌配位酶的关键残基，Asp-104和Asp-143可能是酶与碱性底物的结合位点。

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.