MUTAGENESIS OF PYRIDOXAL PHOSPHATE DEPENDENT ENZYMES

这项资助的主要目的是探索因素n 允许区分底物的酶设计。二 天冬氨酸氨基转移酶和酪氨酸 氨基转移酶(TATase)，两者都将氨基酸转化为2- 含氧酸。前者在衬底选择性方面对 二羧酸，L-天冬氨酸和L-谷氨酸，而后者是 允许和容纳，此外，芳香氨基酸。 特异性由Arg292的侧链控制，即 AATase中的刚性，以阻止芳香氨基酸，但可移动 在TATase中，并允许几乎所有种类的氨基 酸。我们将使用基因工程，结合 动能技术，试图理解 侧链受蛋白质超结构的限制。进一步 基因工程实验将被用来逆转 底物电荷专一性，从而将AATase转化为 精氨酸转氨酶。这将涉及额外的基于结构的 我们研究的9个ARG292 Asp突变的突变 几年前。与结晶学家在 瑞士的目标是对两个新的 磷酸吡哆醛依赖酶，上面讨论的TATase， 和ACC合成酶，这是控制乙烯的关键酶 在工厂里生产。乙烯是一种成熟的植物， 衰老激素。其他目标是1)了解 ACC合成酶机制的能量学细节 确定重要的中间体对反应的影响 路径分配返回到反应物或产品；2)到 确定C抽象的过渡态结构 S-腺苷甲硫氨酸在乙酰胆碱合成酶中的α质子 布朗斯特德分析；3)评估一个不寻常的SO- 氨基酸称为AATase中的低能垒氢键 修改；4)开发一种明确的技术来诊断 存在衬底沟道，从而第N次方的乘积 代谢途径中的酶直接输送到第N+1位 酶，它是底物，而不是首先被释放 变成了溶液。

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.