Postproline cleaving enzyme: identification as serine protease using active site specific inhibitors.
Postproline cleaving enzyme: identification as serine protease using active site specific inhibitors.
复制标题
脯氨酸后裂解酶：使用活性位点特异性抑制剂鉴定为丝氨酸蛋白酶。
DOI：
10.1021/bi00632a022
复制
发表时间：
1977
期刊：
影响因子：
2.9
通讯作者：
R. Walter
中科院分区：
文献类型：
--
作者：
T. Yoshimoto;R. Orlowski;R. Walter
Tadashi Yoshimoto, Ronald C. Orlowski, and Roderich Walter* abstract: Postproline cleaving enzyme (PPCE)(EC 3.4. 21.-) is an endopeptidase which has a strong specificity for catalyzing thecleavage of the Pro-X peptide bond. The en-zyme also exhibits esterase activity. Highly purified PPCE is not inhibited by EDTA or 1, 10-phenanthroline and partial inhibition is obtained only with high molar ratios of p-hy-droxymercuribenzoic acid, sodium tetrathionate, 5, 5'-dithio-bis (2-nitrobenzoic acid), or 2-iodoacetamide. Both peptidase and esterase activities are lost upon treatment with diisopropyl phosphorofluoridate (iPr2P-F). Titration withInitiated inhibitor,[3H] iPr2P-F, reveals that this inhibition is irreversible and occurs on a 1: 1 molar basis. PPCE inhibited by treatment with [3H] iPr2P-F retainsthe p/of 4.8 of unmodifiedPPCE and has a similar subunit molecular weight (58 000) on sodium dodecyl sulfate electrophoresis as unmodified PPCE, and identical chromatographic properties on the affinity column Z-Pro-D-Ala-poly (Lys)-Sepharose 4B. The pH dependence of incorporation of [3H] iPr2P-F is similar to that of the enzy-matic hydrolysis of the standard substrate Z-Gly-Pro-Leu-Gly. í^ stproline cleaving enzyme (PPCE)* 1 was first discovered in human uterus, when it was found that preparations of this target organ of oxytocin cleave the prolyl7-leucyl8 peptide bond of the nonapeptide hormone (Walter et al., 1971). Subsequent studies seem to suggest that this enzymatic activity is ubiqui-tous in vertebrates, but is present only at low levels (Walter, 1973). The enzyme has been purified from lamb kidney and characterized as the first known endopeptidase with a high specificity for cleaving the-L-Pro-X-peptide bond except for the-L.-Pro-L, D-Pro-bond, which for all practical purposes is not hydrolyzed (Walter, 1976; Koida and Walter, 1976). In this paper, information on the active site of PPCE is obtained by inhibition and kinetic studies, and the catalytic mechanisms of both the peptidase and esterase activitiesof the enzyme are investigated. While reagents known to inhibit sulfhydryl proteases (Glazer and Smith, 1971) and metal-l From the Department of Physiology and Biophysics, University of Illinois at the Medical Center, Chicago, Illinois 60612. Received January 12, 1977. Supported by National Institutes of Health Grant AM-18399.
