Savory P J, Djaballah H, Angliker H, Shaw E, Rivett A J
Department of Biochemistry, University of Leicester, U.K.
Biochem J. 1993 Dec 15;296 ( Pt 3)(Pt 3):601-5. doi: 10.1042/bj2960601.
The multicatalytic endopeptidase complex (proteasome) has multiple distinct peptidase activities. These activities have often been referred to as 'chymotrypsin-like', 'trypsin-like' and 'peptidylglutamyl-peptide hydrolase' activities according to the type of residue in the P1 position, although it is now clear that mammalian proteasomes have at least five distinct catalytic sites. In the present study, potential affinity-labelling reagents (peptidylchloromethanes, peptidyldiazomethanes, a peptidylfluoromethane and peptidylsulphonium salts) containing hydrophobic, basic or acidic amino acid residues in the P1 position have been tested for inhibition of the different activities of the rat liver proteinase complex. The results show that individual peptidase activities of proteasomes can be inhibited by a variety of peptidylchloromethanes and peptidyldiazomethanes. Although the rate of inactivation of proteasomes by even the most effective peptidylchloromethanes and peptidyldiazomethanes are often quite slow (k(obs)/[I] in the range 0.1-10 M-1 x s-1) compared with the reaction of similar compounds with some other proteinases, the results provide useful information concerning the specificity of the distinct catalytic centres of proteasomes, and some selective affinity-labelling reagents have been identified. Tyr-Gly-Arg-chloromethane was found to be a useful inhibitor of trypsin-like activity. Inhibition of the other peptidase activities was often incomplete, even after repeated addition of inhibitor, and it proved to be difficult to predict the effect of different reagents. For example, Cbz-Tyr-Ala-Glu-chloromethane was found to inhibit 'chymotrypsin-like' activity (assayed with Ala-Ala-Phe-7-amino-4-methylcoumarin or succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin), while the best inhibitors of 'peptidylglutamyl-peptide hydrolase' activities (assayed with benzyloxycarbonyl-Leu-Leu-Glu beta-naphthylamide) were peptidyldiazomethanes containing hydrophobic amino acid residues. These results suggest that the original nomenclature of proteasome activities is misleading, because the residue in the P1 position is not the only determinant of specificity.
多催化内肽酶复合物（蛋白酶体）具有多种不同的肽酶活性。根据P1位置残基的类型，这些活性通常被称为“类胰凝乳蛋白酶”、“类胰蛋白酶”和“肽基谷氨酰肽水解酶”活性，尽管现在已经清楚哺乳动物蛋白酶体至少有五个不同的催化位点。在本研究中，测试了在P1位置含有疏水、碱性或酸性氨基酸残基的潜在亲和标记试剂（肽基氯甲烷、肽基重氮甲烷、肽基氟甲烷和肽基锍盐）对大鼠肝脏蛋白酶复合物不同活性的抑制作用。结果表明，蛋白酶体的各个肽酶活性可被多种肽基氯甲烷和肽基重氮甲烷抑制。尽管与类似化合物与其他一些蛋白酶的反应相比，即使是最有效的肽基氯甲烷和肽基重氮甲烷使蛋白酶体失活的速率通常也相当缓慢（观测到的速率常数k(obs)/[I]在0.1 - 10 M-1·s-1范围内），但这些结果提供了有关蛋白酶体不同催化中心特异性的有用信息，并且已经鉴定出一些选择性亲和标记试剂。发现酪氨酸 - 甘氨酸 - 精氨酸 - 氯甲烷是类胰蛋白酶活性的有效抑制剂。即使重复添加抑制剂，对其他肽酶活性的抑制通常也不完全，并且事实证明很难预测不同试剂的效果。例如，发现苄氧羰基 - 酪氨酸 - 丙氨酸 - 谷氨酸 - 氯甲烷可抑制“类胰凝乳蛋白酶”活性（用丙氨酸 - 丙氨酸 - 苯丙氨酸 - 7 - 氨基 - 4 - 甲基香豆素或琥珀酰 - 亮氨酸 - 亮氨酸 - 缬氨酸 - 酪氨酸 - 7 - 氨基 - 4 - 甲基香豆素测定），而“肽基谷氨酰肽水解酶”活性（用苄氧羰基 - 亮氨酸 - 亮氨酸 - 谷氨酸β - 萘酰胺测定）的最佳抑制剂是含有疏水氨基酸残基的肽基重氮甲烷。这些结果表明，蛋白酶体活性的原始命名具有误导性，因为P1位置的残基不是特异性的唯一决定因素。
