Griffin Joanna, Engel Paul C
School of Biomolecular and Biomedical Science, Conway Institute, University College Dublin, Belfield, Dublin 4, Ireland.
Enzyme Res. 2011;2011:595793. doi: 10.4061/2011/595793. Epub 2011 Aug 16.
Sequence and structure comparisons of various glutamate dehydrogenases (GDH) and other nicotinamide nucleotide-dependent dehydrogenases have potentially implicated certain residues in coenzyme binding and discrimination. We have mutated key residues in Clostridium symbiosum NAD(+)-specific GDH to investigate their contribution to specificity and to enhance acceptance of NADPH. Comparisons with E. coli NADPH-dependent GDH prompted design of mutants F238S, P262S, and F238S/P262S, which were purified and assessed at pH 6.0, 7.0, and 8.0. They showed markedly increased catalytic efficiency with NADPH, especially at pH 8.0 (∼170-fold for P262S and F238S/P262S with relatively small changes for NADH). A positive charge introduced through the D263K mutation also greatly increased catalytic efficiency with NADPH (over 100-fold at pH 8) and slightly decreased activity with NADH. At position 242, "P6" of the "core fingerprint," where NAD(+)- and NADP(+)-dependent enzymes normally have Gly or Ala, respectively, clostridial GDH already has Ala. Replacement with Gly produced negligible shift in coenzyme specificity.
对各种谷氨酸脱氢酶（GDH）以及其他烟酰胺核苷酸依赖性脱氢酶进行的序列和结构比较，可能表明某些残基在辅酶结合和识别中发挥作用。我们对共生梭菌NAD⁺特异性GDH中的关键残基进行了突变，以研究它们对特异性的贡献，并提高对NADPH的接受度。与大肠杆菌NADPH依赖性GDH的比较促使我们设计了F238S、P262S和F238S/P262S突变体，并在pH 6.0、7.0和8.0条件下对其进行了纯化和评估。它们对NADPH的催化效率显著提高，尤其是在pH 8.0时（P262S和F238S/P262S提高了约170倍，而对NADH的影响相对较小）。通过D263K突变引入的正电荷也大大提高了对NADPH的催化效率（在pH 8时提高了100倍以上），同时使对NADH的活性略有降低。在242位，即“核心指纹”的“P6”位置，NAD⁺依赖性和NADP⁺依赖性酶通常分别具有甘氨酸或丙氨酸，而梭菌GDH已经具有丙氨酸。用甘氨酸替代后，辅酶特异性的变化可忽略不计。
