STRUCTURAL AND ENZYMATIC CHARACTERIZATION OF A PURIFIED PROHORMONE-PROCESSING ENZYME - SECRETED, SOLUBLE KEX2 PROTEASE
STRUCTURAL AND ENZYMATIC CHARACTERIZATION OF A PURIFIED PROHORMONE-PROCESSING ENZYME - SECRETED, SOLUBLE KEX2 PROTEASE
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DOI：
10.1073/pnas.89.3.922
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发表时间：
1992-02-01
影响因子：
11.1
通讯作者：
FULLER, RS
中科院分区：
文献类型：
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作者：
BRENNER, C;FULLER, RS
出芽酵母酿酒酵母的激素加工原酶Kex2蛋白酶可以通过删除跨膜结构域和c末端尾巴，从胞内膜蛋白转化为可溶性、分泌性和活性形式。一个这样的分子被纯化到接近。过表达酵母菌株培养基的同质性。氨基酸序列分析表明，成熟的Kex2蛋白酶的N端是由Lys108-Arg109的潜在自蛋白水解切割产生的，然后是与枯草菌素同源的结构域，然后是由Ste13二肽基氨基肽酶修剪Leu-Pro和Val-Pro二肽。采用荧光肽基甲基香豆素酰胺底物检测动力学参数。初始爆发滴定表明该制剂完全有活性。测定活性对pH值的依赖性，得到一条简单的曲线，表明滴定的是一个可电离基团。活性在pH 5.7时达到一半最大值，在pH 6.5 ~ 9.5时基本保持不变。在K(m)和K（cat）中，不同底物之间的区别高达360倍和130倍。在裂解键之前含有赖氨酸精氨酸二肽的底物是首选的，其k(cat)/ k(m)值高达1.1 X 10(7) sec-1-M-1。该酶对含有Arg-Arg、Pro-Arg、Ala-Arg和Thr-Arg的底物的裂解K(m)增加，但K（cat）不变。相比之下，该酶对lyst，ys底物的k（cat）显著降低，k(m)增加较小。因此，断裂键之前的两个残基可能在激素原底物的结合和切割的选择性中起着不同的作用。
The prohormone-processing Kex2 protease of the budding yeast Saccharomyces cerevisiae can be converted from an intracellular membrane protein to a soluble, secreted, and active form by deletion of the transmembrane domain and C-terminal tail. One such molecule was purified to near. homogeneity from the culture medium of an overexpressing yeast strain. Amino acid sequence analysis revealed that the N terminus of mature Kex2 protease is created by a potentially autoproteolytic cleavage at Lys108-Arg109, prior to the domain homologous to subtilisin, followed by trimming of Leu-Pro and Val-Pro dipeptides by the Ste13 dipeptidyl aminopeptidase. Kinetic parameters were examined using fluorogenic peptidyl-methylcoumarin amide substrates. Initial burst titration indicated that the preparation was entirely active. Measurements of dependence of activity on pH yielded a simple curve suggesting titration of a single ionizable group. Activity was half-maximal at pH 5.7 and nearly constant from pH 6.5 to 9.5. Discrimination between substrates was as great as 360-fold in K(m) and 130-fold in k(cat). Substrates with a Lys-Arg dipeptide preceding the cleaved bond were preferred, having k(cat)/K(m) values up to 1.1 X 10(7) sec-1-M-1. The enzyme cleaved substrates having Arg-Arg, Pro-Arg, Ala-Arg, and Thr-Arg with increased K(m) but with unchanged k(cat). In contrast, the enzyme displayed a dramatically lower k(cat) for a Lys-t,ys substrate with a smaller increase in K(m). Thus the two residues preceding the cleaved bond may play distinct roles in the selectivity of binding and cleavage of prohormone substrates.