STRUCTURE AND FUNCTION OF THE GAMMA-GLUTAMYL CARBOXYLASE

维生素K依赖性γ-谷氨酰羧化酶将其底物蛋白中的谷氨酸后转化为γ羧基谷氨酸（Gla）。这种修饰通过多种维生素K依赖性蛋白对正常血液凝固至关重要，并且通过基质Gla蛋白的参与对钙稳态至关重要。很可能会发现这种翻译后修饰的其他关键作用。在这里，我们探讨的结构和功能的羧化酶。我们已经证明羧化酶将维生素K活化为高活性辅因子中间体的能力受含谷氨酸底物的存在的调节。这种调节至少部分是由于酶中关键的游离半胱氨酸残基的环境的改变。在具体的目标1中，我们确定了机械上重要的半胱氨酸，并确定了羧化酶中的二硫键模式。在具体目标2中，我们确定了内质网膜内羧化酶的拓扑结构。我们将确定酶中被占据的潜在N-连接糖基化位点，进行糖基化扫描诱变，并结合蛋白酶保护试验使用无细胞转录和翻译。 在具体目标3中，我们使用串联质谱的创新方法，在离子碎裂过程中保留γ-羧基，以确定羧化酶是进行性的还是分布性的，如果是分布性的，它是定向的还是随机的？我们开发了一种转基因小鼠模型，其中，在羧化酶基因敲除小鼠的背景下，我们引入了一个野生型羧化酶转基因的肝脏特异性启动子的调控下。这挽救了羧化酶缺失小鼠的胎儿表型。在具体目标4中，我们将使用该模型来识别其他关键的γ-羧基化蛋白质。我们还将使用小鼠模型来确定各种羧化酶底物的动力学参数与这些底物的体内羧化的关系。维生素K依赖性羧化酶进行对血液凝固和钙稳态至关重要的翻译后修饰。提出的研究将阐明羧化酶机制的重要方面，并提供一个拓扑结构，以统一的机制信息已经为这种酶。这些研究的性能将通过该计划的其他方面得到提高：核心服务的可用性、项目II中关于羧化酶基因表达调控的信息以及大卫罗斯和布鲁斯福瑞博士在这些研究的各个方面的技术专长。

The vitamin K-dependent gamma-glutamyl carboxylase post-translationally converts glutamic acids in its substrate proteins to gamma carboxyglutamic acid (Gla). This modification is critical for normal blood coagulation, through multiple vitamin K-dependent proteins, and for calcium homeostasis, through participation of matrix Gla protein. It is likely that other critical roles for this post-translational modification will be discovered. Here we explore the structure and function of the carboxylase. We have been shown that the ability of the carboxylase to activate vitamin K to the highly reactive cofactor intermediate is regulated by the presence of glutamate containing substrate. This regulation results from modification, at least in part, of the environment of a critical free cysteine residue in the enzyme. In specific Aim 1 we identify the mechanistically important cysteine(s) and determine the disulfide bonding pattern in carboxylase. In Specific Aim 2, we determine the topology of the carboxylase within the endoplasmic reticulum membrane. We will determine the potential N-linked glycosylation sites in the enzyme that are occupied, perform glycosylation scanning mutagenesis and use cell free transcription and translation in conjunction with protease protection assays. In Specific Aim 3 we use innovative methods of tandem mass spectrometry that spares gamma-carboxyl groups during ion fragmentation to determine if the carboxylase is processive or distributive, if distributive, is it directional or random? We developed a transgenic mouse model in which, on the background of a carboxylase null mouse, we introduced a wild-type carboxylase transgene under the regulation of a liver specific promoter. This rescues the fetal phenotype of the carboxylase null mouse. In Specific Aim 4 we will use this model to identify additional critical gamma-carboxylated proteins. We will also use the mouse model to determine the relationship of kinetic parameters of various carboxylase substrates to in vivo carboxylation of these substrates. The vitamin K-dependent carboxylase performs a post-translational modification critical for blood coagulation and for calcium homeostasis. The studies proposed will elucidate important aspects of carboxylase mechanism and provide a topologic structure to unify the mechanistic information already available for this enzyme. The performance of these studies will be enhanced by other aspects of the Program: the availability of Core services, information about regulation of carboxylase gene expression from Project II as well as the technical expertise of Drs. David Roth and Bruce Furie in various aspects of these studies.