Analysis of Histidine Decarboxylase Cooperativity by Crystallography and Protein Engineering

通过晶体学和蛋白质工程分析组氨酸脱羧酶协同性

• 批准号: 9601096
• 负责人: Jon Robertus
• 金额: $ 24万
• 依托单位: University of Texas at Austin
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-08-15 至 2000-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9601096&HistoricalAwards=false
• 关键词: Analysis; Histidine; Decarboxylase; Cooperativity; Crystallography

9601096 Robertus Histidine decarboxylase (HDC) from Lactobacillus 30a converts histidine to histamine and CO2. The enzyme shows cooperative regulation. It is activated by low pH. The goal is to examine the mechanism of this activation in atomic detail using X-ray crystallography and site-specific mutagenesis. A kinetic analysis of HDC found the wild-type enzyme can be described as having a Tense state T with low substrate affinity and a Relaxed state R with higher affinity; low pH and high histidine concentrations stabilize the R form. Several site-directed mutants, including some double mutants, have been made which stabilize the T form. The X-ray structure of HDC originally solved by the Hackert group corresponds to the R state at pH 4.8. Two mutants of the enzyme have been crystallized in its T state. X-ray data have been collected to 3.1 ( resolution on one form and molecular replacement and SIR phased solutions of the structures are underway. It is also proposed to complete the molecular structure of several other important mutants which have been crystallized. The T structures will be compare with the known R model to describe tertiary and quaternary structural changes involved in the cooperative mechanism. Residues likely to play prominent roles in the T to R conversion will be identified. A substrate analog, histidine methyl ester HME, will be soaked into the crystals and the mode of substrate binding analyzed. These will be compared with the binding already described for R state HDC at pH 4.8. Difference in binding caused by pH and/or by quaternary structural rearrangement will be elucidated. %%% The overall goal of this research is to shed light on the atomic forces and interactions which turn enzymes on and off. The principles involved in this kind of regulation are universal, and what we learn studying an enzyme from bacteria will help us understand the rules used by all living systems. The switch trigger under study in this project, the simple hydrogen ion, is of considerable importance. The reason is that many metabolic processes involve the movement or production of hydrogen ions, and indeed they are among the most common effectors of biologic activity known. In spite of the simplicity and breadth of this kind of enzyme regulation, there are presently no detailed models of how hydrogen ions turn proteins off or on at the atomic level. We have formed a likely hypothesis that the positively charged ion binds between two molecules of the enzyme when they are arranged in a ring, and "pulls" them into an active conformation. When the hydrogen ion level in the cell decreases, the ones trapped in the protein exit leaving two negative charges at this site on the proteins to repel one another. This repulsion changes the enzyme shape into an inactive conformation. The work in this proposal should produce an atomic model of the enzyme with and without the bound hydrogen ion and allow us to actually see if the hypothesis is correct. This in turn will build our confidence that we can understand the strategies used by other living systems to regulate enzyme activity according to the local hydrogen ion concentration (pH). ***

9601096来自乳杆菌30 a的Robertus组氨酸脱羧酶（HDC）将组氨酸转化为组胺和CO2。 该酶具有协同调节作用。 它是激活的低pH值。我们的目标是检查这种激活的原子细节，使用X射线晶体学和定点诱变的机制。 HDC的动力学分析发现，野生型酶可以被描述为具有低底物亲和力的紧张状态T和具有较高亲和力的松弛状态R;低pH和高组氨酸浓度稳定R形式。 已经制备了几种定点突变体，包括一些双突变体，其稳定了T形式。 最初由Hackert组解决的HDC的X射线结构对应于pH 4.8时的R状态。 该酶的两个突变体已在其T状态下结晶。 X射线数据已经收集到3.1（一种形式的分辨率和分子置换和结构的SIR阶段解决方案正在进行中。 它还建议完成其他几个重要的突变体已结晶的分子结构。 T结构将与已知的R模型进行比较，以描述合作机制中涉及的三级和四级结构变化。 将鉴定可能在T至R转化中起重要作用的残基。 将底物类似物组氨酸甲酯HME浸入晶体中，并分析底物结合模式。 这些将与已经描述的在pH 4.8下R状态HDC的结合进行比较。 将阐明pH和/或四级结构重排引起的结合差异。 这项研究的总体目标是阐明原子力和相互作用，使酶打开和关闭。 这种调节所涉及的原则是普遍的，我们从细菌中研究酶所学到的东西将有助于我们理解所有生命系统所使用的规则。 在这个项目中研究的开关触发器，简单的氢离子，是相当重要的。 原因是许多代谢过程涉及氢离子的运动或产生，事实上，它们是已知生物活性的最常见效应物之一。 尽管这种酶的调节简单而广泛，但目前还没有氢离子如何在原子水平上关闭或打开蛋白质的详细模型。 我们已经形成了一个可能的假设，即当酶的两个分子排列成环时，带正电荷的离子在它们之间结合，并将它们“拉”成活性构象。 当细胞中的氢离子水平降低时，被困在蛋白质中的氢离子退出，在蛋白质上的这个位点留下两个负电荷，以相互排斥。 这种排斥作用将酶的形状改变为非活性构象。 这项提案中的工作应该产生一个有和没有结合氢离子的酶的原子模型，并允许我们实际看看假设是否正确。这反过来将建立我们的信心，我们可以理解其他生命系统根据局部氢离子浓度（pH）调节酶活性的策略。 ***