| Budget Amount *help |
¥3,300,000 (Direct Cost: ¥3,300,000)
Fiscal Year 2004: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 2003: ¥2,400,000 (Direct Cost: ¥2,400,000)
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| Research Abstract |
For L.pentosus D-LDH, replacement of Tyr52 with Leu, Val, and Ala induced size-dependent changes in the specificity of the enzyme to aliphatic or aromatic 2-ketoacid substrates, indicating that the size or shape of hydrophobic side chain at position 52 determines the space of the binding site for hydrophobic side chains of 2-ketoacid substrates. The replacements of Tyr52 with Arg, Thr and Asp, and those of Phe299 with Gly and Ser greatly reduced the enzyme activities toward all 2-ketoacids tested, and induced slow but significant catalysis of NADH oxidation without substrate. However, the double mutations for positions 52 and 299 did not additively injured the enzyme function, but compensated each other for substrate-independent NADH oxidation and catalytic function for some substrates. 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 Asn97Asp mutant
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D-LDH exhibited virtually the same k_<cat>, but about 70-fold higher K_M 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 K_M value, but 110 and 590-fold decreases in the k_<cat> values for formate, respectively. Furthermore, these mutant FDHs, particularly the Glu141Asn enzyme, exhibited markedly enhanced catalytic activity for glyoxylate reduction, indicating that FDH is converted to a 2-hydroxyacid dehydrogenase on the replacement of Glu 141. 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.
For L.casei allosteric L-LDH, which exhibits relatively wide substrate specificity for 2-ketoacids, replacements of Arg173 with Gln and of His188 with Ala or Asp revealed that some 2-ketoacids can be bound to both the catalytic and allosteric sites of the enzyme, and the binding to the allosteric site can exhibit significant activation effects. These replacements also indicated that Arg173 is essential for the 2-ketoacid binding to the allosteric site, and His188 determines the specificity toward 2-ketoacids. Less
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