RUI: Prosthetic Decarboxylase Catalysts

利用位点定向诱变的方法研究了由洋葱假单胞菌（Pseudomonas cepacia）催化的2,2-二烷基甘氨酸脱羧酶（EC 4.1.1.64）（一种吡哆醛5'-磷酸依赖酶，可使2,2-二烷基甘氨酸和d -氨基酸脱羧，但使l -氨基酸转胺）催化的脱羧反应中氨基酸侧链的作用。活性位点组氨酸和赖氨酸侧链的功能将分别通过丙氨酸和甘氨酸取代这些氨基酸，然后使用各种小分子模拟失去的侧链的功能来研究。这些是假体或辅助催化剂。这种方法为活性位点侧链的功能提供了一种直接的测试：如果添加的分子对碱性更强的分子的催化作用更有效，那么原始侧链很可能起着碱的作用。如果添加的分子对碱性较强的分子的催化效率较低，那么原来的侧链可能起酸的作用。Toney和Kirsch首先使用各种小碱基来测试赖氨酸在天冬氨酸转肽酶中的功能ıScience 243, 1485 (1989)！，将用于检测二氯甘氨酸脱羧酶的赖氨酸。将使用一系列咪唑来测试活性组氨酸在同一酶中的功能。

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.