pThe covalent attachment of SUMO (small ubiquitin-like protein modifier) to target proteins results in modifications in their activity, binding interactions, localization or half-life. The reversal of this modification is catalysed by SENPs (SUMO-specific processing proteases). Mammals contain four SUMO paralogues and six SENP enzymes. In the present paper, we describe a systematic analysis of human SENPs, integrating estimates of relative selectivity for SUMO1 and SUMO2, and kinetic measurements of recombinant C-terminal cSENPs (SENP catalytic domains). We first characterized the reaction of each endogenous SENP and cSENPs with HA–SUMO-VS [HA (haemagglutinin)-tagged SUMO-vinyl sulfones], active-site-directed irreversible inhibitors of SENPs. We found that all cSENPs and endogenous SENP1 react with both SUMO paralogues, whereas all other endogeneous SENPs in mammalian cells and tissues display high selectivity for SUMO2-VS. To obtain more quantitative data, the kinetic properties of purified cSENPs were determined using SUMO1- or SUMO2-AMC (7-amino-4-methylcoumarin) as substrate. All enzymes bind their respective substrates with high affinity. cSENP1 and cSENP2 process either SUMO substrate with similar affinity and catalytic efficiency; cSENP5 and cSENP6 show marked catalytic specificity for SUMO2 as measured by iK/isubm/sub and ik/isubcat/sub, whereas cSENP7 works only on SUMO2. Compared with cSENPs, recombinant full-length SENP1 and SENP2 show differences in SUMO selectivity, indicating that paralogue specificity is influenced by the presence of the variable N-terminal domain of each SENP. Our data suggest that SUMO2 metabolism is more dynamic than that of SUMO1 since most SENPs display a marked preference for SUMO2./p
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机译：> SUMO（类小泛素蛋白修饰剂）与目标蛋白的共价结合导致其活性，结合相互作用，定位或半衰期的改变。 SENPs（SUMO特异性加工蛋白酶）催化这种修饰的逆转。哺乳动物包含四个SUMO旁系同源物和六个SENP酶。在本文中，我们描述了人类SENP的系统分析，整合了SUMO1和SUMO2相对选择性的估计以及重组C末端cSENP（SENP催化域）的动力学测量。我们首先表征了每个内源性SENP和cSENPs与SENPs的活性位点导向的不可逆抑制剂HA–SUMO-VS [HA（血凝素）标记的SUMO-乙烯基砜]的反应。我们发现，所有cSENP和内源性SENP1都与SUMO旁系同源物发生反应，而哺乳动物细胞和组织中的所有其他内源性SENP对SUMO2-VS显示出高选择性。为了获得更多的定量数据，使用SUMO1-或SUMO2-AMC（7-氨基-4-甲基香豆素）作为底物测定了纯化的cSENP的动力学性质。所有酶都以高亲和力结合其各自的底物。 cSENP1和cSENP2处理具有相似亲和力和催化效率的SUMO底物; cSENP5和cSENP6对SUMO2表现出明显的催化特异性，通过 K m 和 k cat 测量，而cSENP7仅起作用在SUMO2上。与cSENPs相比，重组全长SENP1和SENP2在SUMO选择性上表现出差异，表明旁系同源特异性受每个SENP可变N端结构域的影响。我们的数据表明，SUMO2的代谢比SUMO1更动态，因为大多数SENP对SUMO2表现出明显的偏爱。
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