Mechanism of Histidinol Dehydrogenase

这项更新提议将继续正在进行的沙门氏菌组氨酸二醇脱氢酶（HDH）机制的研究，这是一种4电子nad1连接酶。重点是利用初级同位素效应来获得两个反应步骤的速率，步骤的化学性质，以及所涉及的酶残基的pK值和性质。高度优先考虑完成脱氢反应的立体化学反应。虽然已经确定了组氨酸与HDH在咪唑环和氨基区域的重要结合相互作用，但这两个基团在空间上的相对位置尚不清楚。刚性组胺受体激动剂将用于确定这一地形。为了确定参与结合或催化的活性位点残基，将使用位点定向修饰和诱变。正在合成逻辑修饰符。除了HDH固有的酶机制外，该系统在生物学中具有广泛的重要性。这种酶是由沙门氏菌组氨酸操纵子的hisD基因编码的，这是一个被特别研究过的遗传系统。系统中已知的突变体构成了众所周知的Ames测试的基础。

This renewal proposal will continue ongoing studies on the mechanism of Salmonella histidinol dehydrogenase (HDH), a 4- electron NAD-linked enzyme. The focus is on the use of primary isotope effects to obtain rates for the two reaction steps, the chemistry of the steps, and the pK values and nature of the enzyme residues involved. A high priority is assigned to completing the stereochemistry of the dehydrogenation reactions. Although important binding interactions of histidinol with HDH at the imidozole ring and amino regions have been identified, the relative positions of these two groups in space are not known. Rigid histamine receptor agonists will be used to determine this topography. In order to identify the active site residues involved in binding or catalysis, site directed modification and mutagenesis will be used. Logical modifiers are being synthesized. In addition to the inherent enzyme mechanistic interest in HDH, the system is of broad importance in biology. The enzyme is coded for by the hisD gene of the Salmonella histidine operon, a particularly well studied genetic system. The known mutants in the system form the basis for the well known Ames test.