METALLOENZYMES AND CONNECTIVE TISSUE METABOLISM

主要蛋白质组分的代谢周转率 细胞外基质由一种叫做基质的酶家族催化。 金属蛋白酶(MMPs)。这个基因家族的两个成员，人类 成纤维细胞胶原酶(HFC)和人中性粒细胞胶原酶(HNC) 唯一能够水解型纤维蛋白I、II和 在生理条件下，胶原蛋白的形成速度相当快。他们 要以非常具体和有特色的方式做到这一点，请制作一个单一的 跨原胶原(TC)单体的所有三条链以 从N-末端大约3/4处产生的特异、敏感的基因座 TCA和TCB片段。这一反应是最具特异性的反应之一 在酶学中，它是胶原蛋白周转的关键步骤。目标是 我们研究的重点之一就是阐明这一非凡现象的潜在基础 从胶原酶的角度对分子识别系统进行研究 胶原酶在胶原蛋白中的裂解部位。HNC的样品将是 准备进行晶体生长实验，使两种晶体的结构 酶原(Prohnc)和活性酶(Hnc)可通过x射线确定。 结晶学。重组酶原(r-Pro-HNC)在E. 并分离出作为x射线用非糖基化酶的来源。 结晶学研究突变的r-Pro-HNC物种将用 自溶激活和降解位置的改变序列以 在晶体中使用的浓度下提高酶的稳定性 生长实验。HFC和HNC的血凝蛋白样域的作用 在确定它们的底物特异性时，将通过携带 在这两者之间进行了一系列的域切换实验 胶原酶，以及它们与其他基质金属蛋白酶之间的相互作用。有关的信息 胶原酶在胶原蛋白中的裂解部位将通过制备一种 固相肽系列模型三螺旋多肽底物 综合。HFC和HNC在递增水平模型上的作用 将研究与胶原酶裂解位点的相似性，以便 阐明本网站的哪些功能对于识别和 被人类胶原酶切割。模型的结晶学研究 HFC和HNC识别和切割最多的三螺旋多肽 将紧密模仿天然胶原蛋白底物进行 以阐明裂解位点的局部结构。

The metabolic turnover of the major protein constituents of the extracellular matrix is catalyzed by a family of enzymes called the matrix metalloproteinases (MMP). Two members of this gene family, human fibroblast collagenase (HFC) and human neutrophil collagenase (HNC), are the only human enzymes capable of hydrolyzing the fibrillar type I, II and III collagens at an appreciable rate under physiological conditions. They do so in a highly specific and characteristic manner by making a single scission across all three chains of the tropocollagen (TC) monomers at a specific, sensitive locus approximately 3/4 from the N-terminus to produce TCA and TCB fragments. This reaction, which is one of the most specific known in enzymology, is the committed step in collagen turnover. The goal of our studies is to elucidate the underlying basis for this remarkable molecular recognition system from the point of view of both the collagenase and the collagenase cleavage site in the collagens. Samples of HNC will be prepared for crystal growth experiments so that the structure of both the zymogen (pro-HNC) and active enzyme (HNC) can be elucidated by x-ray crystallography. Recombinant zymogen (r-pro-HNC) will be expressed in E. coli and isolated as a source of unglycosylated enzyme for x-ray crystallographic studies. Mutant r-pro-HNC species will be prepared with altered sequences at the autolytic activation and degradation sites to improve the stability of the enzyme at the concentrations used in crystal growth experiments. The role of the hemopexin-like domain of HFC and HNC in determining their substrate specificity will be investigated by carrying out a series of domain switching experiments between these two collagenases, as well as between them and other MMP. Information about the collagenase cleavage site in collagens will be investigated by preparing a series of model, triple helical peptide substrates by solid phase peptide synthesis. The action of HFC and HNC on models with ascending levels of resemblance to the collagenase cleavage site will be studied in order to elucidate features of this site that are critical for recognition and cleavage by human collagenases. Crystallographic studies of the model triple helical peptides whose recognition and cleavage by HFC and HNC most closely mimic that of the natural collagen substrates will be carried out to elucidate the local structure of the cleavage site.