MUTAGENESIS OF PYRIDOXAL PHOSPHATE DEPENDENT ENZYMES

这项补助金的主要目的是探讨因素n 酶设计允许底物之间的区别。 两 天冬氨酸转氨酶（AAT酶）和酪氨酸 氨基转移酶（TATase），两者都将氨基酸转化为2- 含氧酸 前者在底物选择性方面对后者是限制性的。 二羧酸氨基酸，L-Asp和L-Glu，而后者是 允许的，并容纳，另外，芳香族氨基酸。 特异性由Arg 292的侧链控制， 在AAT酶中是刚性的，以阻止芳香族氨基酸进入，但是是移动的 在TATase，并允许接近几乎所有种类的氨基 acids. 我们将使用基因工程， 动力学技术，试图了解 侧链受到蛋白质超结构的限制。 进一步 基因工程实验将被用来逆转 底物电荷特异性，并因此将AAT酶转化为 精氨酸转氨酶 这将涉及额外的基于结构的 ARG 292 Asp突变，我们调查了9个 年前 与晶体学家合作， 瑞士被指示对两个新的 磷酸吡哆醛依赖性酶，上述TATase， ACC合成酶是控制乙烯的关键酶 在工厂生产。 乙烯是植物成熟和 衰老激素 额外的目标是1）了解 ACC合酶机制的能量学细节， 确定反应中的重要中间体 路径分配回到反应物或产物上; 2） 确定用于C的抽象的过渡状态结构 ACC合酶中S-腺苷甲硫氨酸的α质子 布朗斯台德分析; 3）评估一个不寻常的如此的意义- AAT酶中的低势垒氢键 修改; 4）开发一种确定的技术来诊断 衬底沟道的存在，由此第N次的产物 代谢途径中的酶被直接递送到第N +1个 酶，它是底物，而不是被释放第一 变成溶液。

The principal objective of this grant is to explore the factors n enzyme design that allow discrimination between substrates. Two enzymes, aspartate aminotransferase (AATase) and tyrosine aminotransferase (TATase), both convert amino acids into 2- oxoacids. The Former is restrictive in substrate selectivity to the dicarboxylic amino acids, L-Asp and L-Glu, while the latter is permissive and accommodates, additionally, aromatic amino acids. Specificity is controlled by the side-chain of Arg292, which is rigid in AATase to keep out aromatic amino acids, but is mobile in TATase, and permits access of nearly all varieties of amino acids. We will use genetic engineering, in combination with kinetic techniques, to try to understand how the mobility of the side chain is restricted by the protein superstructure. Further genetic engineering experiments will be employed to reverse substrate charge specificity, and thus, convert AATase to an arginine transaminase. This will involve additional structure-based mutations of the ARG292 Asp mutation that we investigated nine years ago. Collaborative work with crystallographers in Switzerland is directed to structure elucidation of two new pyridoxal phosphate dependent enzymes, TATase discussed above, and ACC synthase, the key enzyme controlling ethylene production in plants. Ethylene is the plant ripening and senescence hormone. Additional goals are 1) to understand the details of the energetics of the ACC synthase mechanism by determining how the important intermediate on the reaction pathway partitions back to reactants or on to products; 2) to determine the transition state structure for the abstraction of the C alpha proton of S-adenosyl methionine in ACC synthase by Bronsted analysis; 3) to evaluate the significance of an unusual so- called, low barrier hydrogen bond in AATase by amino acid modification; 4) to develop a definitive technique to diagnose the existence of substrate channeling, whereby the product of the Nth enzyme in a metabolic pathway is delivered directly to the Nth+1 enzyme, for which it is a substrate, rather than being released first into solution.