Microbial carboxyl proteinases related to a fatal neurodegenerative disease: proposal for a novel catalytic mechanism

我们对微生物胃抑素不敏感的羧基蛋白酶(PSCP、XSCP、Kumamolisin和J-4)及其哺乳动物同源物、与致死性神经退行性疾病相关的CLN2基因产物(CLN2蛋白)进行了酶和分子生物学研究。将这些结果与PSCP和Kumamolisin的结构分析结果相比较，阐明了它们的结构和新的催化机理，并讨论了它们的分子进化。2001年的总结：(I)保守的丝氨酸残基通过定点突变研究了五种酶(PSCP、XSCP、kumamolisin、J-4和CLN2蛋白)之间的保守丝氨酸残基。(Ii)PSCP的结构分析及其催化机理分析表明，由Ser287、GIu80和Asp84残基组成的新型催化三联体参与了其催化功能。经…鉴定为PSCP的催化残基。设计合成了新型抑制剂，并将其用于熊果菌素的结构分析。2002年总结：(I)前-PSCPS287A突变体的结构分析表达为不溶的前-原蛋白，复性后的酶不具有任何自催化加工和蛋白酶活性。这些结果有力地表明Ser287残基参与了催化残基的形成。S287A突变体的结构分析正在进行中。(Ii)熊果素中的催化残基根据熊果素的结构，Glu32和Trp129残基被认为是熊果素分子中唯一的催化残基。突变株E32A和W129A的K_&lt；cat&gt；/K_m分别为对照的6%和4%，表明这两个残基都参与了催化作用。(3)CLN_2蛋白的结构分析经动力学分析，CLN_2蛋白有一个底物结合部位，由六个亚基(S3-S3‘)组成。对家蚕蛹中表达的CLN2蛋白的结构分析正在进行中。较少

We carrited out enzymatic and molecular biological investigations on microbial pepstatin-insensitive carboxyl proteinases (PSCP, XSCP, kumamolisin, and J-4) and their mammalian homologue, the CLN2 gene product (CLN2 protein), which is related to the fatal neurodegenerative disease. Compared these results with the data from structural analyses of PSCP and kumamolisin, we clarified their structures and novel catalytic mechanism and discussed on their molecular evolution, too.SUMMARY IN 2001:(i) Conserved serine residuesThe conserved serine residues among five enzymes (PSCP, XSCP, kumamolisin, J-4, and the CLN2 protein) were elucidated to be involved in the catalytic residues by their site-directed mutagenesis studies.(ii) Structural analysis of PSCP and its catalytic mechanismStructural analysis has revealed that a novel type of catalytic triad composed of Ser287, GIu80, and Asp84 residues is involved in its catalytic function. They were confirmed to be the catalytic residues of PSCP by … More site-directed mutagenesis and inhibitor studies.(iii) New inhibitors for kumamolisin and its applicationNovel type of inhibitors were designed and synthesized, and they were used for structural analysis of kumamolisin.SUMMARY IN 2002:(i) Structural analysis of prepro-PSCPS287A mutant was expressed as the insoluble prepro-protein, and the refolded enzyme did not show any autocatalytic processing- and proteinase-activity. These results strongly suggested that Ser287 residue was involved in the catalytic residues. Structural analysis of the S287A mutant is now in progress.(ii) Catalytic residues in kumamolisinBased on the structure of kumamolisin, Glu32 and Trp129 residues were suggested to be an unique catalytic residues in kumamolisin molecule. The k_<cat>/K_m values of the E32A and W129A mutants were about 6 and 4 % of that of the control, indicating that both residues were involved in the catalytic function.(iii) Structural analysis of the CLN2 proteinThe CLN2 protein was elucidated to have a substrate-binding site consisting of six subsites (S3-S3') by kinetics analysis. Structural analysis of the CLN2 protein expressed in silkworm pupae is now in progress. Less