METALLOBIOCHEMISTRY OF BONE MORPHOGENETIC PROTEIN 1

骨形态发生蛋白 1 的金属生物化学

• 批准号: 2023088
• 负责人: DAVID S AULD
• 金额: $ 16.03万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-01-01 至 1998-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2023088
• 关键词: active sites; chemical binding; chemical kinetics; conformation; enzyme activity; enzyme inhibitors; enzyme substrate; high performance liquid chromatography; metalloendopeptidases; molecular site; peptide chemical synthesis; protein sequence; site directed mutagenesis; stromelysin; zymogens

Metalloproteinases are active in hormone processing, digestion and in many situations requiring remodeling of the extracellular matrix, .g. normal morphogenesis, wound healing and uterine resorption. Long bone formation, which depends on bone morphogenetic factors, e.g. BMP-1, also likely involves metalloproteinase action. We recently identified a zinc protease, astacin, by recognizing a potential catalytic zinc binding site signature sequence, HExxHxxGxxH. This signature differs from that seen in thermolysin and carboxypeptidase A and an x-ray structure of astacin now confirms our prediction that the 3 histidines are ligands to the zinc. The signature occurs in 60 different proteins which can be subdivided by homology into the astacin, snake venom, g-negative bacterium and matrix metalloproteinase (MMP) families. Recent x-ray crystallographic structures of a member of each of these sub-families also confirms the presence of this zinc binding site. The astacin family consists of a group of proteins that have a 200 amino acid domain that is homologous to astacin. These include human, mouse and Xenopus laevis BMP-1, the mouse and rat kidney and human intestinal brush border metalloendopeptidases, the protein encoded by the Drosophila dorsal-ventral patterning gene tolloid and the proteins encoded by one avian, two sea urchin and three fish embryogenesis genes. The smallest member of the MMP family, pump-1, contains the basic features for both latent and active enzyme forms of this family. It has the propeptide domain that contains a single Cys believed to be involved in zymogen activation and the central domain that contains the zinc signature. It has both the catalytic zinc binding site and a second, possible structural zinc site. Several reactive amino acids are conserved in both the astacin family and the MMP family. The present study addresses the chemical properties of two of the immediate family members, astacin and BMP-1 and two of the extended family members of the MMPs pump-1 and the catalytic domain of stromelysin-1, strom-1. A combination of kinetic and spectroscopic approaches that allow direct visualization of ES complexes and site-directed mutagenic changes of the enzymes will examine a) the substrate specificity, b) the characteristics of the mechanism, c) reveal the amino acids that are critical for substrate binding and catalysis, d) activation of latent forms. The results of this study will be used to design assays based on specific substrates and inhibitors for the detection of these enzymes under physiological and early developmental cellular conditions.

金属蛋白酶在激素加工、消化和许多疾病中具有活性。 需要细胞外基质重塑的情况，例如，正常 形态发生、伤口愈合和子宫吸收。 长骨形成， 这取决于骨形态发生因子，如BMP-1，也可能 涉及金属蛋白酶作用。 我们最近发现了一种锌蛋白酶， 通过识别潜在的催化锌结合位点标记， 序列，HExxHxxGxxH。 这一特征不同于 嗜热菌蛋白酶和羧肽酶A以及虾红素的X射线结构 证实了我们的预测，即3个组氨酸是锌的配体。 的 标签存在于60种不同的蛋白质中，这些蛋白质可以细分为 同源性分为虾青素、蛇毒、G-阴性菌和基质 金属蛋白酶（MMP）家族。 最近的X射线晶体结构 这些子家族中的每一个成员的存在也证实了 这个锌结合位点。 astacin家族由一组蛋白质组成 其具有与虾红素同源200个氨基酸的结构域。 这些 包括人、小鼠和非洲爪蟾BMP-1，小鼠和大鼠肾， 人肠刷状缘金属内肽酶，由 果蝇背腹模式基因tolloid及其蛋白质 由一个鸟类、两个海胆和三个鱼类胚胎发生基因编码。 MMP系列中最小的成员，泵-1，包含基本功能 对于这个家族的潜在和活性酶形式。 它有 前肽结构域，含有一个Cys，据信参与 酶原激活和含有锌标记的中心结构域。 它既有催化锌结合位点， 结构锌位点。 几个反应性氨基酸是保守的， astacin家族和MMP家族。 本研究报告讨论了 两个直系亲属的化学性质， BMP-1和MMPs泵-1和MMPs的两个扩展家族成员， strom-1的催化结构域。 动力学和动力学的结合 光谱方法，允许直接可视化ES复合物 和酶的定点诱变变化将检查a） 底物特异性，B）机制的特征，c）揭示 对底物结合和催化至关重要的氨基酸，d） 激活潜在形式。 本研究的结果将用于 基于特定底物和抑制剂设计检测方法 这些酶在生理和早期发育细胞 条件