Distinct Conformation-mediated Functions of an Active Site Loop in the Catalytic Reactions of NAD-dependent D-Lactate Dehydrogenase and Formate Dehydrogenase*
Distinct Conformation-mediated Functions of an Active Site Loop in the Catalytic Reactions of NAD-dependent D-Lactate Dehydrogenase and Formate Dehydrogenase*
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DOI：
10.1074/jbc.m500970200
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发表时间：
2005-04
影响因子：
4.8
通讯作者：
Takeshi Shinoda;Kazuhito Arai;Mayu Shigematsu-Iida;Yoshirou Ishikura;Satoru Tanaka;Takashi Yamada;M. Kimber;E. Pai;S. Fushinobu;H. Taguchi
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文献类型：
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作者：
Takeshi Shinoda;Kazuhito Arai;Mayu Shigematsu-Iida;Yoshirou Ishikura;Satoru Tanaka;Takashi Yamada;M. Kimber;E. Pai;S. Fushinobu;H. Taguchi
nadd依赖性d-乳酸脱氢酶（d-LDH）和甲酸脱氢酶（FDH）的三维结构相似，这意味着这两种酶通常使用某些主链原子，这些原子位于两种酶活性位点的相应环结构上，以实现各自的催化功能。这些活性位点环在两种酶中采用不同的构象，可能是由于与Asn97和Glu141的氢键，它们也分别位于d-LDH和FDH的等效位置。2.4-Å分辨率的x射线晶体学显示，Asp替代Asn97并没有显著改变整体蛋白结构，但明显扰乱了戊酸乳杆菌d-LDH活性位点环的构象。Asn97→Asp突变体d-LDH表现出几乎相同的kcat，但对丙酮酸的KM值比野生型高约70倍。对于副球菌sp. 12-A FDH，用Gln和Asn替代Glu141仅诱导KM值增加5.5倍和4.3倍，而甲酸的kcat值分别降低110倍和590倍。此外，这些突变体FDH，特别是Glu141→Asn酶，对乙醛酸还原的催化活性显著增强，表明FDH在取代Glu141后转化为2-羟基酸脱氢酶。这些结果表明，活性位点环分别在d-LDH和FDH的催化反应、稳定底物结合和促进氢转移中发挥着不同的作用，而Asn97和Glu141稳定了合适的环构象，是环正常功能的必要元素。
The three-dimensional structures of NAD-dependent d-lactate dehydrogenase (d-LDH) and formate dehydrogenase (FDH), which resemble each other, imply that the two enzymes commonly employ certain main chain atoms, which are located on corresponding loop structures in the active sites of the two enzymes, for their respective catalytic functions. These active site loops adopt different conformations in the two enzymes, a difference likely attributable to hydrogen bonds with Asn97 and Glu141, which are also located at equivalent positions in d-LDH and FDH, respectively. X-ray crystallography at 2.4-Å resolution revealed that replacement of Asn97 with Asp did not markedly change the overall protein structure but markedly perturbed the conformation of the active site loop in Lactobacillus pentosus d-LDH. The Asn97 → Asp mutant d-LDH exhibited virtually the same kcat, but about 70-fold higher KM value for pyruvate than the wild-type enzyme. For Paracoccus sp. 12-A FDH, in contrast, replacement of Glu141 with Gln and Asn induced only 5.5- and 4.3-fold increases in the KM value, but 110 and 590-fold decreases in the kcat values for formate, respectively. Furthermore, these mutant FDHs, particularly the Glu141 → Asn enzyme, exhibited markedly enhanced catalytic activity for glyoxylate reduction, indicating that FDH is converted to a 2-hydroxy-acid dehydrogenase on the replacement of Glu141. These results indicate that the active site loops play different roles in the catalytic reactions of d-LDH and FDH, stabilization of substrate binding and promotion of hydrogen transfer, respectively, and that Asn97 and Glu141, which stabilize suitable loop conformations, are essential elements for proper loop functioning.