METALLOENZYMES AND CONNECTIVE TISSUE METABOLISM

金属酶和结缔组织代谢

• 批准号: 3275184
• 负责人: HAROLD E VAN WART
• 金额: $ 12.23万
• 依托单位: SYNTEX (USA), INC.-RESEARCH DIVISION
• 依托单位国家: 美国
• 财政年份: 1980
• 资助国家: 美国
• 起止时间: 1980-04-01 至 1996-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3275184
• 关键词: Escherichia coli; SDS polyacrylamide gel electrophoresis; X ray crystallography; active sites; chemical cleavage; chemical kinetics; chemical models; chimeric proteins; collagenase; connective tissue metabolism; crystallization; enzyme activity; enzyme mechanism; enzyme structure; enzyme substrate; enzyme substrate analog; metalloenzyme; mutant; peptide chemical synthesis; protein degradation; recombinant DNA; western blottings; zymogens

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.

的主要蛋白质成分的代谢周转， 细胞外基质是由称为基质的酶家族催化的 金属蛋白酶（MMP）。 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）， 唯一能够水解纤维状I型、II型和III型蛋白质的人类酶， III胶原蛋白在生理条件下以可观的速率。 他们 这样做，在一个高度具体和特点的方式，使一个单一的 原胶原（TC）单体的所有三条链在 特异性的，敏感的位点，大约3/4的N-末端，以产生 TCA和TCB片段。 这种反应是最具特异性的 是胶原蛋白周转的关键步骤。 目标 我们的研究的重点是阐明这一非凡现象的潜在基础 分子识别系统从胶原酶的角度 和胶原中的胶原酶切割位点。 HNC样品将 准备晶体生长实验，使结构的两个 酶原（pro-HNC）和活性酶（HNC）可通过X射线解析 结晶学 重组酶原（r-pro-HNC）在大肠杆菌中表达。 大肠杆菌，并作为X射线的非糖基化酶的来源分离 晶体学研究 将使用以下物质制备突变体r-pro-HNC物质： 改变自溶活化和降解位点的序列， 在晶体中使用的浓度下提高酶的稳定性 生长实验 血红蛋白样结构域在HFC和HNC中的作用 在确定其底物特异性时，将通过进行 我们做了一系列这两者之间的域转换实验， 胶原酶，以及它们与其他MMP之间的关系。 信息 胶原蛋白中的胶原酶切割位点将通过制备 一系列模型，三螺旋肽底物通过固相肽 合成. HFC和HNC对具有上升水平的模型的作用 将研究与胶原酶切割位点的相似性， 阐明该网站的特点，是识别的关键， 通过人胶原酶切割。 模型的晶体学研究 三螺旋肽，其被HFC和HNC的识别和切割最 将进行紧密模拟天然胶原基质的实验 以阐明切割位点的局部结构。