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首页> 外文期刊>Biological chemistry >Biochemical characterization of the catalytic domains of three different clostridial collagenases.
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Biochemical characterization of the catalytic domains of three different clostridial collagenases.

机译：三种不同梭菌胶原酶催化域的生化特征。

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摘要

Clostridial collagenases are used for a broad spectrum of biotechnological applications and represent prime target candidates for both therapy and diagnosis of clostridial infections. In this study, we biochemically characterized the catalytic domains of three clostridial collagenases, collagenase G (ColG) and H (ColH) from Clostridium histolyticum, and collagenase T (ColT) from C. tetani. All protein samples showed activity against a synthetic peptidic substrate (furylacryloyl-Leu-Gly-Pro-Ala, FALGPA) with ColH showing the highest overall activity and highest substrate affinity. Whereas the K(m) values of all three enzymes were within the same order of magnitude, the turnover rate k(cat) of ColG decreased 50- to 150-fold when compared to ColT and ColH. It is noteworthy that the protein N-terminus significantly impacts their substrate affinity and substrate turnover as well as their inhibition profile with 1,10-phenanthroline. These findings were complemented with the discovery of a strictly conserved double-glycine motif, positioned 28 amino acids upstream of the HEXXH zinc binding site, which is critical for enzymatic activity. These observations have consequences with respect to the topology of the N-terminus relative to the active site as well as possible activation mechanisms.
机译：梭菌胶原酶被广泛应用于生物技术领域，是梭菌感染治疗和诊断的主要靶标候选物。在这项研究中，我们对三种梭菌胶原酶的催化域进行了生化表征，这三种酶分别是溶组织梭状芽胞杆菌的胶原酶G（ColG）和H（ColH）和破伤风梭菌的胶原酶T（ColT）。所有蛋白质样品均显示出对合成肽底物（呋喃基丙烯酰基-Leu-Gly-Pro-Ala，FALGPA）的活性，ColH显示出最高的总体活性和最高的底物亲和力。尽管所有三种酶的K（m）值都在同一数量级内，但与ColT和ColH相比，ColG的周转率k（cat）降低了50-150倍。值得注意的是，蛋白质N末端会显着影响其底物亲和力和底物周转率以及它们对1,10-菲咯啉的抑制作用。这些发现与严格保守的双甘氨酸基序的发现相辅相成，该基序位于HEXXH锌结合位点上游28个氨基酸处，这对酶促活性至关重要。这些观察结果对N末端相对于活性位点的拓扑结构以及可能的激活机制具有影响。

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