Mechanism of Histidinol Dehydrogenase

这项更新提案将继续正在进行的关于沙门氏菌组氨酸脱氢酶(HDH)机制的研究，HDH是一种4电子NAD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，以及所涉及的酶残留物的pK值和性质。高度优先考虑的是完成脱氢反应的立体化学。虽然组氨醇与HDH在咪唑环和氨基区域的重要结合作用已被确定，但这两个基团在空间上的相对位置尚不清楚。硬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，将使用定点修饰和突变。逻辑修饰语正在被合成。除了对HDH固有的酶机制感兴趣外，该系统在生物学上也具有广泛的重要性。这种酶是由组氨酸沙门氏菌操纵子的HISD基因编码的，这是一个研究特别充分的遗传系统。该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.