Analysis of Histidine Decarboxylase Cooperativity by Crystallography and Protein Engineering

来自乳杆菌30a的Robertus Histidine decarboxylase （HDC）可将组氨酸转化为组胺和CO2。酶表现出协同调节。它被低ph激活。目标是使用x射线晶体学和位点特异性诱变在原子细节上检查这种激活的机制。HDC的动力学分析发现，野生型酶具有低底物亲和力的紧张状态T和高亲和力的放松状态R；低pH和高组氨酸浓度稳定R型。一些位点定向突变体，包括一些双突变体，已被制成稳定T型。最初由Hackert基团解析的HDC的x射线结构对应于pH值为4.8时的R态。该酶的两个突变体已在其T状态下结晶。x射线数据已收集到一种形式的3.1（分辨率），分子替代和SIR相解的结构正在进行中。还建议完成其他几个已结晶的重要突变体的分子结构。将T结构与已知的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。将确定可能在T到R转化中起突出作用的残基。将底物类似物组氨酸甲酯HME浸泡在晶体中，分析底物结合模式。这些将与已经描述的pH值为4.8的R态HDC的结合进行比较。由pH和/或由四级结构重排引起的结合差异将被阐明。这项研究的总体目标是阐明原子力和使酶开启和关闭的相互作用。这种调节所涉及的原则是普遍的，我们从细菌中学习的一种酶将帮助我们理解所有生命系统使用的规则。本课题所研究的开关触发器——简单氢离子具有相当重要的意义。原因是许多代谢过程涉及氢离子的运动或产生，事实上，它们是已知的生物活性中最常见的效应器之一。尽管这种酶的调节简单而广泛，但目前还没有关于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，即当两个酶分子排列成一个环时，带正电的离子结合在酶分子之间，并将它们“拉”成一个活跃的构象。当细胞中的氢离子水平降低时，被困在蛋白质中的氢离子就会离开，在蛋白质的这个位置留下两个负电荷，彼此排斥。这种斥力使酶的形状变成无活性的构象。这个提议中的工作应该会产生一个有和没有氢离子结合的酶的原子模型，并允许我们实际看看假设是否正确。反过来，这将建立我们的信心，我们可以了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。＊＊＊

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). ***