RUI: Prosthetic Decarboxylase Catalysts

洋葱假单胞菌2，2-二甘氨酸脱羧酸酯(EC4.1.1.64)是一种依赖5‘-磷酸的吡哆醛脱羧酶，能脱羧基2，2-二甘氨酸和D-氨基酸，转氨酶L-氨基酸，研究氨基酸侧链在该酶催化的脱羧基反应中的作用。活性部位组氨酸和赖氨酸侧链的功能将通过分别用丙氨酸和甘氨酸取代这些氨基酸，然后使用各种小分子来模拟丢失的侧链的功能来研究。这些是假体或辅助催化剂。这种方法为活性中心侧链的功能提供了直接的测试：如果添加的分子对更基本的分子的催化往往更有效，那么原来的侧链可能起到碱基的作用。如果添加的分子对更碱性的分子的催化效率较低，那么原来的侧链可能起到酸的作用。Toney和Kirsch首次使用各种小碱基来测试天冬氨酸转氨酶ı2431485(1989)中赖氨酸的功能，这些碱基将用于测试二甘氨酸脱羧酶的赖氨酸。一系列咪唑类化合物将用于测试同一酶中活性组氨酸的功能。

The role of amino acid side chains in decarboxylation reactions catalyzed by Pseudomonas cepacia 2,2-dialklglycine decarboxylates (EC 4.1.1.64), a pyridoxal 5'-phosphate-dependent enzyme which decarboxylates 2,2-dialklglycines and D-amino acids but transaminates L-amino acids, will be investigated using site directed mutagenesis. The function of active site histidine and lysine side chains will be studied by replacing these amino acids by alanine and glycine, respectively, and then using various small molecules to mimic the function of the lost side chains. These are prosthetic or auxiliary catalysts. This approach provides a direct test for the function of an active site side chain: If catalysis by the added molecules tends to be more efficient with molecules that are more basic, then the original side chain is likely to function as a base. If catalysis by the added molecules tends to be less efficient with molecules that are more basic, then the original side chain likely functions as an acid. A variety of small bases, first used by Toney and Kirsch to test the function of a lysine in aspartate aminotransferase ıScience 243, 1485 (1989)!, will be used to test the lysine of the dialklglycine decarboxylase. A series of imidazoles will be used to test the function of the active histidine in the same enzyme.