Håkansson Anders P, Smith Alexander W
Department of Microbiology and Immunology, University at Buffalo, State University of New York, Buffalo, New York 14214, USA.
J Biol Chem. 2007 Oct 5;282(40):29521-30. doi: 10.1074/jbc.M703144200. Epub 2007 Aug 9.
This study describes the enzymatic characterization of dihydrolipoamide dehydrogenase (DLDH) from Streptococcus pneumoniae and is the first characterization of a DLDH that carries its own substrate (a lipoic acid covalently attached to a lipoyl protein domain) within its own sequence. Full-length recombinant DLDH (rDLDH) was expressed and compared with enzyme expressed in the absence of lipoic acid (rDLDH(-LA)) or with enzyme lacking the first 112 amino acids constituting the lipoyl protein domain (rDLDH(-LIPOYL)). All three proteins contained 1 mol of FAD/mol of protein, had a higher activity for the conversion of NAD(+) to NADH than for the reaction in the reverse direction, and were unable to use NADP(+) and NADPH as substrates. The enzymes had similar substrate specificities, with the K(m) for NAD(+) being approximately 20 times higher than that for dihydrolipoamide. The kinetic pattern suggested a Ping Pong Bi Bi mechanism, which was verified by product inhibition studies. The protein expressed without lipoic acid was indistinguishable from the wild-type protein in all analyses. On the other hand, the protein without a lipoyl protein domain had a 2-3-fold higher turnover number, a lower K(I) for NADH, and a higher K(I) for lipoamide compared with the other two enzymes. The results suggest that the lipoyl protein domain (but not lipoic acid alone) plays a regulatory role in the enzymatic characteristics of pneumococcal DLDH.
本研究描述了肺炎链球菌二氢硫辛酰胺脱氢酶（DLDH）的酶学特性，这是首次对在其自身序列中携带自身底物（共价连接至硫辛酰蛋白结构域的硫辛酸）的DLDH进行特性描述。表达了全长重组DLDH（rDLDH），并将其与在无硫辛酸情况下表达的酶（rDLDH(-LA)）或缺少构成硫辛酰蛋白结构域的前112个氨基酸的酶（rDLDH(-LIPOYL)）进行比较。所有这三种蛋白质每摩尔蛋白质均含有1摩尔FAD，将NAD(+)转化为NADH的活性高于其逆向反应活性，并且无法将NADP(+)和NADPH用作底物。这些酶具有相似的底物特异性，NAD(+)的K(m)约为二氢硫辛酰胺的20倍。动力学模式表明为乒乓双双机制，这通过产物抑制研究得到了验证。在所有分析中，无硫辛酸表达的蛋白质与野生型蛋白质无差异。另一方面，与其他两种酶相比，没有硫辛酰蛋白结构域的蛋白质具有高2 - 3倍的周转数、较低的NADH的K(I)和较高的硫辛酰胺的K(I)。结果表明，硫辛酰蛋白结构域（而非单独的硫辛酸）在肺炎球菌DLDH的酶学特性中起调节作用。
