Carrigan John B, Engel Paul C
School of Biomolecular and Biomedical Science, Conway Institute, University College Dublin, Ireland.
FEBS J. 2007 Oct;274(19):5167-74. doi: 10.1111/j.1742-4658.2007.06038.x. Epub 2007 Sep 10.
Glutamate dehydrogenase (EC 1.4.1.2-4) from Peptostreptococcus asaccharolyticus has a strong preference for NADH over NADPH as a coenzyme, over 1000-fold in terms of kcat/Km values. Sequence alignments across the wider family of NAD(P)-dependent dehydrogenases might suggest that this preference is mainly due to a negatively charged glutamate at position 243 (E243) in the adenine ribose-binding pocket. We have examined the possibility of altering coenzyme specificity of the Peptostreptococcus enzyme, and, more specifically, the role of residue 243 and neighbouring residues in coenzyme binding, by introducing a range of point mutations. Glutamate dehydrogenases are unusual among dehydrogenases in that NADPH-specific forms usually have aspartate at this position. However, replacement of E243 with aspartate led to only a nine-fold relaxation of the strong discrimination against NADPH. By contrast, replacement with a more positively charged lysine or arginine, as found in NADPH-dependent members of other dehydrogenase families, allows a more than 1000-fold shift toward NADPH, resulting in enzymes equally efficient with NADH or NADPH. Smaller shifts in the same direction were also observed in enzymes where a neighboring tryptophan, W244, was replaced by a smaller alanine (approximately six-fold) or Asp245 was changed to lysine (32-fold). Coenzyme binding studies confirm that the mutations result in the expected major changes in relative affinities for NADH and NADPH, and pH studies indicate that improved affinity for the extra phosphate of NADPH is the predominant reason for the increased catalytic efficiency with this coenzyme. The marked difference between the results of replacing E243 with aspartate and with positive residues implies that the mode of NADPH binding in naturally occurring NADPH-dependent glutamate dehydrogenases differs from that adopted in E243K or E243D and in other dehydrogenases.
来自不解糖消化链球菌的谷氨酸脱氢酶（EC 1.4.1.2 - 4）作为辅酶，对NADH的偏好远高于NADPH，就kcat/Km值而言，这种偏好超过1000倍。对更广泛的NAD(P)依赖性脱氢酶家族进行序列比对可能表明，这种偏好主要是由于腺嘌呤核糖结合口袋中第243位的带负电荷的谷氨酸（E243）。我们通过引入一系列点突变，研究了改变不解糖消化链球菌酶辅酶特异性的可能性，更具体地说，是研究243位残基及相邻残基在辅酶结合中的作用。谷氨酸脱氢酶在脱氢酶中不同寻常，因为NADPH特异性形式在此位置通常为天冬氨酸。然而，用天冬氨酸取代E243仅导致对NADPH的强烈歧视放松了9倍。相比之下，用其他脱氢酶家族中依赖NADPH的成员中发现的带更多正电荷的赖氨酸或精氨酸取代，会使对NADPH的偏好发生超过1000倍的转变，从而产生对NADH或NADPH效率相同的酶。在邻近的色氨酸W244被较小的丙氨酸取代（约6倍）或Asp245变为赖氨酸（32倍）的酶中，也观察到了相同方向的较小转变。辅酶结合研究证实，这些突变导致了对NADH和NADPH相对亲和力的预期重大变化，pH研究表明，对NADPH额外磷酸基团亲和力的提高是该辅酶催化效率增加的主要原因。用天冬氨酸和正性残基取代E243的结果之间的显著差异意味着，天然存在的依赖NADPH的谷氨酸脱氢酶中NADPH的结合模式与E243K或E243D以及其他脱氢酶中采用的模式不同。
