Distinct Conformation-mediated Functions of an Active Site Loop in the Catalytic Reactions of NAD-dependent D-Lactate Dehydrogenase and Formate Dehydrogenase

• Published in: The Journal of biological chemistry 2005-04, Vol.280 (17), p.17068-17075
• Main Authors: Shinoda, Takeshi, Arai, Kazuhito, Shigematsu-Iida, Mayu, Ishikura, Yoshirou, Tanaka, Satoru, Yamada, Takashi, Kimber, Matthew S., Pai, Emil F., Fushinobu, Shinya, Taguchi, Hayao
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Asparagine - chemistry; Base Sequence; Binding Sites; Catalytic Domain; Crystallography, X-Ray; Dose-Response Relationship, Drug; Formate Dehydrogenases - chemistry; Glutamic Acid - chemistry; Hydrogen Bonding; Kinetics; Lactate Dehydrogenases - chemistry; Lactic Acid - chemistry; Lactobacillus - enzymology; Models, Chemical; Models, Molecular; Molecular Sequence Data; Mutagenesis, Site-Directed; Mutation; NADP - chemistry; Oxygen - chemistry; Paracoccus - enzymology; Protein Conformation; Sequence Homology, Amino Acid; Substrate Specificity; Thermodynamics
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M500970200

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 pentosusd-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.