Pepstatin-Insensitive Carboxyl Proteinase : Glutamic Proteinase

众所周知，羧基蛋白酶通常受到pepstatin^<1)>、DAN^<2)>和EPNP^<3)>的抑制，它们的催化残基由两个天冬氨酸残基组成。因此，羧基蛋白酶被称为天冬氨酸蛋白酶。这些酶在一级和三级结构上都高度同源。我们已经从真菌、细菌和嗜热细菌中分离出了新的羧基蛋白酶，这些酶对胃抑素、DAN和EPNP不敏感。这些酶暂定名为胃抑素不敏感羧基蛋白酶。在我们的一项研究中，从真菌Scytalidium lignicolum中建立了羧基蛋白酶B（由204个氨基酸组成）的一级结构，其中一个催化残基被澄清为Glu-53。这是关于谷氨酸蛋白酶的首次报道。似乎胃抑素不敏感的羧基蛋白酶不是天冬氨酸蛋白酶，而是谷氨酸蛋白酶。为了证实这种可能性，我们研究了从假单胞菌sp. No. 101中分离到的一种对胃抑素不敏感的羧基蛋白酶，这是第一个从原核细胞中分离到的羧基蛋白酶。我们确定了酶的初级结构是一个由363个氨基酸残基和一个二硫桥组成的单一多肽。所鉴定的一级结构与目前报道的天冬氨酸蛋白酶没有任何同源结构。此外，在天冬氨酸蛋白酶的活性中心没有观察到保守的- asp *- thr - gly -结构。为了将抑制剂应用于活性中心的研究，我们从北桃孢菌（Kitasatosporia sp. 55）中分离出一种新的抑制剂。鉴定为n -异valeryl- tyroyl -leucyl-tyrosinal，命名为tyrostatin。酪氨酸抑素对假单胞菌101号抑素不敏感的羧基蛋白酶具有化学计量抑制作用。利用酪氨酸抑素或其衍生物鉴定羧基假单胞菌蛋白酶催化残基的进一步研究正在进行中。1)胃抑素，胃蛋白酶抑制剂；2) DAN，重氮乙酰- dl -去甲亮氨酸甲基lester；3) EPNP, 1,2-epou-3-（对硝基苯氧基）丙烷。少

It is well known that carboxyl proteases are commonly inhibited by pepstatin^<1)> , DAN^<2)> and EPNP^<3)> , and their catalytic residues are composed of two aspartic residues. Thus, carboxyl proteases are termed aspartic proteases. These enzymes are highly homologous in both the primary and tertiary structures.We have isolated novel carboxyl proteases from fungi, bacteria and also thermophilic bacteria based on their insensitivities to pepstatin, DAN and EPNP. These enzymes were tentatively named pepstatin-insensitve carboxyl proteases. In one of our studies, the primary structure of carboxyl protease B ( consisting of 204 aminb acids) from a fungus Scytalidium lignicolum has been established, one of the catalytic residues of which was clarified to be Glu-53. This is the first report on glutamic protease.It seemed probable that the pepstatin-insensitive carboxyl proteases are not aspartic proteases but glutamic proteases. To confirm this possibility, we studied a pepstatin-insensitive … More carboxyl protease from Pseudomonas sp. No. 101, which is the the first carboxyl protease isolated from prokaryote cells. We determined the primary structure of the enzyme as a single polypeptide composed of 363 amino acid residues with one disulfide bridge. The elucidated primary structure does not have any homologous structure to those of aspartic proteases reported so far. Moreover, the well-conserved structure, -Asp*-Thr-Gly- in the active center of aspartic proteases was not observed.In our attempt to use inhibitor in the study of active center we isolated a novel inhibitor from Kitasatosporia sp. No. 55. It was identified as N-isovaleryl-tyrosyl-leucyl-tyrosinal and named it tyrostatin. Tyrostatin was found to inhibit stoichiometrically pepstatin- insensitive carboxyl proteinases of Pseuomonas sp. No. 101.Further study on identification of the catalytic residues of Pseudomonas carboxyl protease using tyrostatin or its derivatives is now in progress. 1 ) pepstatin, pepsin inhibitor; 2) DAN, diazoacetyl-DL-norleucine methylester; 3) EPNP, 1,2-epou-3-(p- nitrophenoxy) propane. Less