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首页> 外文期刊>Biochemistry >Improved specificity toward substrates with positively charged side chains by site-directed mutagenesis of the L-lactate dehydrogenase of Bacillus stearothermophilus.
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Improved specificity toward substrates with positively charged side chains by site-directed mutagenesis of the L-lactate dehydrogenase of Bacillus stearothermophilus.

机译：通过对嗜热脂肪芽孢杆菌的L-乳酸脱氢酶进行定点诱变，提高了对带有正电荷侧链的底物的特异性。

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摘要

The substrate specificities of L-alpha-hydroxy acid dehydrogenases, including L-lactate dehydrogenases (L-LDH's), can often be quite broad. However, an LDH with high catalytic activity toward alpha-keto acids with positively charged side chains, such as those containing ammonium groups, has not been described, even though there is evidence from metabolic studies that a natural dehydrogenase with such activity might exist in Nature. L-omega-Amino-alpha-hydroxy acids are important intermediates in the synthesis of pharmacologically active compounds, and enzymatic reduction of omega-amino-alpha-keto acids represents an attractive route to these compounds. Graphics analysis indicated that introduction of acidic amino acids at position 102 of the L-LDH of Bacillus stearothermophilus (BSLDH) would favor binding of such side chain ammonium groups. Accordingly, Q102E and Q102D mutant BSLDH's were constructed and the steady state kinetic parameters determined for these mutants for a broad range of alpha-keto acids, including omega-amino-keto acids. The results obtained show that, compared to WT-BSLDH, these mutants show up to 25-fold improvements in kcat/Km values for omega-amino-alpha-keto acid substrates.
机译：L-α-羟酸脱氢酶，包括L-乳酸脱氢酶（L-LDH's）的底物特异性通常很宽。然而，尽管有代谢研究的证据表明在自然界中可能存在具有这种活性的天然脱氢酶，但LDH对具有带正电侧链的α-酮酸（例如含有铵基的侧链）具有高催化活性。 。 L-ω-氨基-α-羟基酸是合成药理活性化合物的重要中间体，ω-氨基-α-酮酸的酶促还原代表了通往这些化合物的诱人途径。图形分析表明在嗜热脂肪芽孢杆菌（BSLDH）的L-LDH的102位引入酸性氨基酸将有利于这种侧链铵基的结合。因此，构建了Q102E和Q102D突变体BSDLH，并确定了这些突变体对于多种α-酮酸（包括ω-氨基-酮酸）的稳态动力学参数。获得的结果表明，与WT-BSLDH相比，这些突变体的ω-氨基-α-酮酸底物的kcat / Km值提高了25倍。

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