Analysis and alteration of the substrate recognition machinery of stereospecific 2-hydroxyacid dehydrogenases from lactic acid bacteria.

对于L.pentosus D-LDH，用Leu、Val和Ala替代Tyr52诱导了酶对脂肪族或芳香族2-酮酸底物特异性的大小依赖性变化，这表明52位疏水侧链的大小或形状决定了2-酮酸底物疏水侧链结合位点的空间。Tyr52被Arg、Thr和Asp取代，Phe299被Gly和Ser取代，大大降低了对所有2-酮酸的酶活性，并诱导了缓慢但显著的无底物催化NADH氧化。然而，52位和299位的双突变并没有对酶的功能造成损伤，而是相互补偿了与底物无关的NADH氧化和对某些底物的催化功能。用Asp替代Asn97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。Asn97Asp突变体…More D-LDH对丙酮酸的K_M值比野生型高约70倍，但K_M值与野生型基本相同。对于副球菌sp.12-A FDH，用Gln和Asn替代Glu141仅诱导K_M值增加5.5倍和4.3倍，而甲酸的k_<cat>值分别降低110倍和590倍。此外，这些突变体FDH，特别是Glu141Asn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。对于L.casei变构L-LDH，它对2-酮酸具有相对广泛的底物特异性，用Gln取代Arg173，用Ala或Asp取代His188，表明一些2-酮酸可以结合在酶的催化位点和变构位点上，并且与变构位点的结合可以表现出明显的激活作用。这些替换也表明Arg173是2-酮酸与变构位点结合所必需的，His188决定了2-酮酸的特异性。少

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 … More 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