METALLOBIOCHEMISTRY OF BONE MORPHOGENETIC PROTEIN 1

骨形态发生蛋白 1 的金属生物化学

• 批准号: 2192604
• 负责人: DAVID S AULD
• 金额: $ 14.95万
• 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 1997-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2192604
• 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.

金属蛋白酶在荷尔蒙的加工、消化和许多 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，例如BMP-1，也可能 涉及到金属蛋白酶的作用。我们最近鉴定了一种锌蛋白酶， 阿司匹林，通过识别潜在的催化锌结合位点特征 序列，HExxHxxGxxH。此签名与中的签名不同 热裂解素和羧基肽酶A及其X-射线结构 证实了我们的预测，即这3个组氨酸是锌的配体。这个 签名出现在60种不同的蛋白质中，这些蛋白质可以细分为 与阿司他星、蛇毒、G-阴性菌和基质同源 金属蛋白酶(MMPs)家族。最新的X射线晶体结构 这些亚科中每一个的成员也证实了 这个锌结合部位。阿司匹林家族由一组蛋白质组成。 它有一个200个氨基酸的结构域，与阿司匹林同源。这些 包括人、小鼠和非洲爪哇BMP-1，小鼠和大鼠肾脏和 人类肠道刷状边界金属内肽酶，编码的蛋白质 果蝇背腹花纹基因tolloid及其蛋白 由一个禽类、两个海胆和三个鱼类胚胎发生基因编码。 基质金属蛋白酶家族中最小的成员，Pump-1，包含基本特征 对于这个家族的潜伏酶和活性酶都适用。它有一个 含有单个半胱氨酸的前肽结构域，据信与 酶原激活和包含锌信号的中心结构域。 它既有催化性的锌结合部位，也有可能的第二个结合部位 结构锌场地。两边都有几种活性氨基酸是保守的。 阿司匹林家族和基质金属蛋白酶家族。本研究涉及 阿司匹林和阿司匹林两个直系亲属的化学性质 BMP-1和MMPs Pump-1和MMPs的两个扩展家族成员 基质分解蛋白-1的催化结构域，Strom-1。动力和动力的结合 允许直接显示ES络合物的光谱方法 而酶的定点突变变化将检查a) 底物专一性，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) 潜伏形式的激活。这项研究的结果将用于 设计基于特定底物和抑制剂的检测方法 这些酶在生理和早期发育细胞中的作用 条件。