Analysis of Histidine Decarboxylase Cooperativity by Crystallography and Protein Engineering

9601096乳杆菌30a产生的罗伯特斯组氨酸脱羧酶能将组氨酸转化为组胺和二氧化碳。该酶表现出协同调节作用。它在低pH值下被激活。目标是利用X射线结晶学和定点突变技术在原子细节上研究这种激活的机制。HDC的动力学分析发现，野生型酶可以描述为具有较低底物亲和力的紧张状态T和亲和力较高的松弛状态R；低pH和高组氨酸浓度稳定R型。一些定点突变体，包括一些双突变体，已经被制备成稳定T型的突变体。HDC的X射线结构最初由Hackert基团解算，对应于pH为4.8时的R态。该酶的两个突变体已结晶为T状态。X射线数据已收集到3.1(一种形式的分辨率)，结构的分子替换和SIR阶段性解决方案正在进行中。还建议完成其他几个重要突变体的分子结构，这些突变体已经结晶。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。将确定在T到R转化过程中可能发挥重要作用的残基。将底物类似物组氨酸甲酯HME浸泡到晶体中，分析底物结合方式。这些将与已经描述的在pH 4.8的R状态HDC的结合进行比较。将阐明由pH和/或四元结构重排引起的结合差异。这项研究的总体目标是阐明启动和关闭酶的原子力和相互作用。这种调节涉及的原理是普遍的，我们从细菌中研究一种酶将有助于我们理解所有生命系统使用的规则。这个项目中正在研究的开关触发器--简单氢离子--是相当重要的。原因是许多代谢过程涉及氢离子的移动或产生，事实上，它们是已知的最常见的生物活动影响因素之一。尽管这种酶的调节简单而广泛，但目前还没有详细的模型来说明氢离子如何在原子水平上关闭或打开蛋白质。我们已经形成了一个可能的假设，即当两个分子排列成环时，带正电的离子结合在酶的两个分子之间，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会在蛋白质的这个位置留下两个负电荷，从而相互排斥。这种排斥力将酶的形状改变为不活跃的构象。这项提议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，并使我们能够真正看到这个假设是否正确。这反过来将建立我们的信心，即我们可以理解其他生命系统根据当地氢离子浓度(PH)来调节酶活性的策略。***

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). ***