Studies on the molecular properties and the structure-function relationships of carboxyl proteases of novel type

1. 用常规的蛋白质化学方法测定了黑曲霉（Aspergillus niger var. macrosporus）产生的一种新型碳蛋白酶（Proctase a）的完整氨基酸序列。此外，还克隆了Proctase A的基因和cDNA，并对其编码区的碱基序列进行了分析。Proctase A对各种蛋白质和肽底物的特异性已经被阐明，活性位点残基已经被化学修饰探索。此外，原proctase A cDNA在大肠杆菌中表达，在酸性条件下具有自催化活性。Proctase A可以结晶，并对其进行了初步的x射线研究。另一方面，二维核磁共振和圆二色性研究表明Proctase A在分子中具有高β -片结构和刚性核结构。Proctase A已被证明在pH值6-7时快速且不可逆地变性，同时两条肽链解离。一种独特的胃抑素不敏感酸性蛋白酶（M 54K）已经从一种真正的黏菌——多头绒泡菌的疟原虫中纯化出来，并显示出独特的双链结构和底物特异性。从人胃粘膜中分离纯化了原athepsin E，明确了其nh_2末端氨基酸序列和碳水化合物附着位点。此外，athepsin E原已被证明具有自催化活性，并且在中性pH下具有显著的蛋白水解活性，而底物特异性较窄。果蝇蛋白酶基因可以在大肠杆菌中表达，产生一种活性酶。它对前体蛋白的切割特异性已被阐明，并且在假定的活性位点上的天冬氨酸残基的活性的重要性已被位点定向诱变证明。

1. The complete amino acid sequence of a carbowxl protease of novel type (Proctase A) produced by the fungus, Aspergillus niger var. macrosporus, has been determined by conventional methods of protein chemistry. Further, the gene and cDNA of Proctase A have been cloned and the base sequence of the coding region of the cDNA has been elucidated.2. The substrate specificity toward various protein and peptide substrates of Proctase A has been elucidated and the active-site residues have been explored by chemical modification. In addition, the cDNA for proproctase A could be expressed in E. coli and shown to be autocatalytically activated under acidic conditions.3. Proctase A could be crystallized and a preliminary X-ray-study on it has been performed. On the other hand, two-dimensional NMR and circular dichroism studies have revealed that Proctase A has a high beta-sheet structure and a rigid core structure in the molecule.4. Proctase A has been shown to be denatured rapidly and irreversibly at pH 6-7 with simultaneous dissociation of the two peptide chains.5. A unique pepstatin-insensitive acid protease (M 54K) has been purified from the plasmodia of a true slime mold, Physarum polycephalum, and shown to have a unique two chain structure and substrate specificity.6. Procathepsin E has been purified from the gastric mucosa of human stomach, and its NH_2-terminal amino acid sequence and the site of carbohydrate attachment have been clarified. Further, procathepsin E has been shown to be autocatalytically activated and to have significant proteolytic activity at neutral pH with rather narrower substrate specificity.7. The gene for Drosophila copia protease could be expressed in E. coli, giving an active enzyme. Its cleavage specificity toward the precursor protein has been elucidated, and the importance for activity of an aspartic acid residue at the putative active site has been demonstrated by site-directed mutagenesis.