Determinants of Substrate Specificity for Saccharopine Dehydrogenase from Saccharomyces cerevisiae
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- 作者：Hengyu Xu ; Ann H. West ; Paul F. Cook
- 刊名：Biochemistry
- 出版年：2007
- 出版时间：June 26, 2007
- 年：2007
- 卷：46
- 期：25
- 页码：7625 - 7636
- 全文大小：172K
- 年卷期：v.46,no.25(June 26, 2007)
- ISSN：1520-4995
文摘
A survey of NADH, 
-Kg, and lysine analogues has been undertaken in an attempt to definethe substrate specificity of saccharopine dehydrogenase and to identify functional groups on all substratesand dinucleotides important for substrate binding. A number of NAD analogues, including NADP,3-acetylpyridine adenine dinucleotide (3-APAD), 3-pyridinealdehyde adenine dinucleotide (3-PAAD), andthionicotinamide adenine dinucleotide (thio-NAD), can serve as a substrate in the oxidative deaminationreaction, as can a number of -keto analogues, including glyoxylate, pyruvate, -ketobutyrate,-ketovalerate, -ketomalonate, and -ketoadipate. Inhibition studies using nucleotide analogues suggestthat the majority of the binding energy of the dinucleotides comes from the AMP portion and that distinctlydifferent conformations are generated upon binding of the oxidized and reduced dinucleotides. Additionof the 2'-phosphate as in NADPH causes poor binding of subsequent substrates but has little effect oncoenzyme binding and catalysis. In addition, the 10-fold decrease in affinity of 3-APAD in comparisonto NAD suggests that the nicotinamide ring binding pocket is hydrophilic. Extensive inhibition studiesusing aliphatic and aromatic keto acid analogues have been carried out to gain insight into the keto acidbinding pocket. Data suggest that a side chain with three carbons (from the -keto group up to and includingthe side chain carboxylate) is optimal. In addition, the distance between the C1-C2 unit and the C5carboxylate of the -keto acid is also important for binding; the -oxo group contributes a factor of 10to affinity. The keto acid binding pocket is relatively large and flexible and can accommodate the bulkyaromatic 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 portionof the amino acid carbon side chain is three or four carbons. In addition, the amino acid binding pocketcan accommodate a branch at the 
-carbon, but not at the 
-carbon.
-Kg, and lysine analogues has been undertaken in an attempt to definethe substrate specificity of saccharopine dehydrogenase and to identify functional groups on all substratesand dinucleotides important for substrate binding. A number of NAD analogues, including NADP,3-acetylpyridine adenine dinucleotide (3-APAD), 3-pyridinealdehyde adenine dinucleotide (3-PAAD), andthionicotinamide adenine dinucleotide (thio-NAD), can serve as a substrate in the oxidative deaminationreaction, as can a number of -keto analogues, including glyoxylate, pyruvate, -ketobutyrate,-ketovalerate, -ketomalonate, and -ketoadipate. Inhibition studies using nucleotide analogues suggestthat the majority of the binding energy of the dinucleotides comes from the AMP portion and that distinctlydifferent conformations are generated upon binding of the oxidized and reduced dinucleotides. Additionof the 2'-phosphate as in NADPH causes poor binding of subsequent substrates but has little effect oncoenzyme binding and catalysis. In addition, the 10-fold decrease in affinity of 3-APAD in comparisonto NAD suggests that the nicotinamide ring binding pocket is hydrophilic. Extensive inhibition studiesusing aliphatic and aromatic keto acid analogues have been carried out to gain insight into the keto acidbinding pocket. Data suggest that a side chain with three carbons (from the -keto group up to and includingthe side chain carboxylate) is optimal. In addition, the distance between the C1-C2 unit and the C5carboxylate of the -keto acid is also important for binding; the -oxo group contributes a factor of 10to affinity. The keto acid binding pocket is relatively large and flexible and can accommodate the bulkyaromatic 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 portionof the amino acid carbon side chain is three or four carbons. In addition, the amino acid binding pocketcan accommodate a branch at the -carbon, but not at the -carbon.