Determinants of substrate specificity for saccharopine dehydrogenase from Saccharomyces cerevisiae.
Determinants of substrate specificity for saccharopine dehydrogenase from Saccharomyces cerevisiae.
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DOI：
10.1021/bi700269p
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发表时间：
2007-06
期刊：
影响因子：
2.9
通讯作者：
Hengyu Xu;A. West;P. Cook
中科院分区：
文献类型：
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作者：
Hengyu Xu;A. West;P. Cook
研究人员对NADH、α - kg和赖氨酸类似物进行了调查，试图确定糖精脱氢酶的底物特异性，并确定所有底物上的官能团和对底物结合重要的二核苷酸。许多NAD类似物，包括NADP、3-乙酰吡啶腺嘌呤二核苷酸（3-APAD）、3-吡啶醛腺嘌呤二核苷酸（3-PAAD）和硫代烟碱酰胺腺嘌呤二核苷酸（thio-NAD），可以作为氧化脱胺反应的底物，许多α -酮类类似物，包括乙醛酸盐、丙酮酸盐、α -酮丁酸盐、α -酮戊酸盐、α -酮丙酸盐和α -酮己二酸盐。使用核苷酸类似物的抑制研究表明，二核苷酸的大部分结合能来自AMP部分，氧化和还原的二核苷酸结合时产生明显不同的构象。NADPH中2'-磷酸的加入导致后续底物的结合较差，但对辅酶的结合和催化作用影响不大。此外，与NAD相比，3-APAD的亲和力降低了10倍，这表明烟酰胺环结合袋具有亲水性。广泛的抑制研究使用脂肪族和芳香酮酸类似物已经进行了深入了解酮酸结合袋。数据表明，具有三个碳的侧链（从α -酮基团到并包括羧酸侧链）是最佳的。此外，α -酮酸的C1-C2单元与C5羧酸之间的距离对结合也很重要；-氧基对亲和力的贡献是10倍。酮酸结合袋较大且灵活，可容纳庞大的吡啶二羧酸芳香环和C3而非C4位置的负电荷。然而，氨基酸结合位点是疏水的，氨基酸碳侧链疏水部分的最佳长度为3或4个碳。此外，氨基酸结合袋可以容纳-碳上的分支，但不能容纳-碳上的分支。
A survey of NADH, alpha-Kg, and lysine analogues has been undertaken in an attempt to define the substrate specificity of saccharopine dehydrogenase and to identify functional groups on all substrates and dinucleotides important for substrate binding. A number of NAD analogues, including NADP, 3-acetylpyridine adenine dinucleotide (3-APAD), 3-pyridinealdehyde adenine dinucleotide (3-PAAD), and thionicotinamide adenine dinucleotide (thio-NAD), can serve as a substrate in the oxidative deamination reaction, as can a number of alpha-keto analogues, including glyoxylate, pyruvate, alpha-ketobutyrate, alpha-ketovalerate, alpha-ketomalonate, and alpha-ketoadipate. Inhibition studies using nucleotide analogues suggest that the majority of the binding energy of the dinucleotides comes from the AMP portion and that distinctly different conformations are generated upon binding of the oxidized and reduced dinucleotides. Addition of the 2'-phosphate as in NADPH causes poor binding of subsequent substrates but has little effect on coenzyme binding and catalysis. In addition, the 10-fold decrease in affinity of 3-APAD in comparison to NAD suggests that the nicotinamide ring binding pocket is hydrophilic. Extensive inhibition studies using aliphatic and aromatic keto acid analogues have been carried out to gain insight into the keto acid binding pocket. Data suggest that a side chain with three carbons (from the alpha-keto group up to and including the side chain carboxylate) is optimal. In addition, the distance between the C1-C2 unit and the C5 carboxylate of the alpha-keto acid is also important for binding; the alpha-oxo group contributes a factor of 10 to affinity. The keto acid binding pocket is relatively large and flexible and can accommodate the bulky aromatic ring of a pyridine dicarboxylic acid and a negative charge at the C3 but not the C4 position. However, the amino acid binding site is hydrophobic, and the optimal length of the hydrophobic portion of the amino acid carbon side chain is three or four carbons. In addition, the amino acid binding pocket can accommodate a branch at the gamma-carbon, but not at the beta-carbon.