Overall kinetic mechanism of saccharopine dehydrogenase (L-glutamate forming) from Saccharomyces cerevisiae.
Overall kinetic mechanism of saccharopine dehydrogenase (L-glutamate forming) from Saccharomyces cerevisiae.
复制标题
酿酒酵母糖碱脱氢酶（L-谷氨酸形成）的总体动力学机制。
DOI：
10.1021/bi800086g
复制
发表时间：
2008
期刊：
影响因子：
2.9
通讯作者：
Cook,PaulF
中科院分区：
文献类型：
--
作者：
Vashishtha,AshwaniKumar;West,AnnH;Cook,PaulF
在pH 7.0条件下，对酿酒酵母中组氨酸标记的糖精还原酶进行了动力学研究，结果表明，在游离酶上有序加入还原后的烟酰胺腺嘌呤二核苷酸磷酸（NADPH），然后再加入-α-氨基己二酸-δ-半醛（l-AASA），在正向反应方向上先加入快速平衡的谷氨酸。在逆反应方向上，向酶中加入烟酰胺腺嘌呤二核苷酸磷酸（NADP），然后加入糖精。NADP对NADPH的抑制是竞争性的，而对α-AASA和l-谷氨酸的抑制是非竞争性的，这表明二核苷酸在醛之前加入了游离酶。糖精对NADPH、α-AASA和l-谷氨酸是非竞争性的。在糖精氧化的方向上，NADPH对NADP是竞争性的，对糖精是非竞争性的，l-谷氨酸对NADP和糖精都是非竞争性的，而- aasa对糖精是非竞争性的，对NADP也是非竞争性的。这一顺序机制也通过使用AASA、l-谷氨酸和糖精的类似物进行的终端抑制研究得到证实。选择2-氨基-6-庚酸作为l-AASA的末端类似物，对AASA具有竞争性，对NADPH无竞争性，对谷氨酸无竞争性。α-酮戊二酸（α-Kg）是l-谷氨酸的末端类似物，对l-谷氨酸具有竞争性，对L-AASA和NADPH无竞争性。在糖精氧化方向上，n -草基甘氨酸、l-胡椒果酸、l-亮氨酸、α-酮戊二酸、乙醛酸和l-鸟氨酸作为糖精的末端类似物，对糖精表现出竞争性抑制，对NADP表现出非竞争性抑制。通过监测NADPH吸光度的变化，在pH 7.0时测定反应的平衡常数为200 M−1。该值与使用霍尔丹关系确定的值一致。
Kinetic studies were carried out for histidine-tagged saccharopine reductase from Saccharomyces cerevisiae at pH 7.0, suggesting a sequential mechanism with ordered addition of reduced nicotinamide adenine dinucleotide phosphate (NADPH) to the free enzyme followed byl-α-aminoadipate-δ-semialdehyde (l-AASA) which adds in rapid equilibrium prior tol-glutamate in the forward reaction direction. In the reverse reaction direction, nicotinamide adenine dinucleotide phosphate (NADP) adds to the enzyme followed by addition of saccharopine. Product inhibition by NADP is competitive vs NADPH and noncompetitive vs α-AASA andl-glutamate, suggesting that the dinucleotide adds to the free enzyme prior to the aldehyde. Saccharopine is noncompetitive vs NADPH, α-AASA, andl-glutamate. In the direction of saccharopine oxidation, NADPH is competitive vs NADP and noncompetitive vs saccharopine,l-glutamate is noncompetitive vs both NADP and saccharopine, whilel-AASA is noncompetitive vs saccharopine and uncompetitive vs NADP. The sequential mechanism is also corroborated by dead-end inhibition studies using analogues of AASA,l-glutamate, and saccharopine. 2-Amino-6-heptenoic acid was chosen as a dead-end analogue ofl-AASA and is competitive vs AASA, uncompetitive vs NADPH, and noncompetitive vsl-glutamate. α-Ketoglutarate (α-Kg) serves as the dead-end analogue ofl-glutamate and is competitive vsl-glutamate and uncompetitive vs L-AASA and NADPH. In the direction of saccharopine oxidation,N-oxalylglycine,l-pipecolic acid,l-leucine, α-ketoglutarate, glyoxylic acid, andl-ornithine were used as dead-end analogues of saccharopine and showed competitive inhibition vs saccharopine and uncompetitive inhibition vs NADP. The equilibrium constant for the reaction was measured at pH 7.0 by monitoring the change in absorbance of NADPH and is 200 M−1. The value is in good agreement with the value determined using the Haldane relationship.