Chemical Mechanism of Saccharopine Reductase from Saccharomyces cerevisiae
Chemical Mechanism of Saccharopine Reductase from Saccharomyces cerevisiae
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DOI：
10.1021/bi900599s
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发表时间：
2009-06-30
期刊：
影响因子：
2.9
通讯作者：
Cook, Paul F.
中科院分区：
文献类型：
--
作者：
Vashishtha, Ashwani Kumar;West, Ann H.;Cook, Paul F.
甜菜碱还原酶（SR）[糖豆碱脱氢酶（L-谷氨酸形成），EC 1.5.1.10]催化L-α-氨基己二酸-δ-半醛（AASA）与L-谷氨酸缩合，生成胺，胺被NADPH还原，生成糖豆碱。酸碱化学机制已被提出SR的基础上的pH值-速率曲线和溶剂氘动力学同位素效应。观察到有限溶剂同位素效应，表明质子在限速步骤中飞行，并且可能相同的步骤在限制和饱和底物浓度下都是限制性的。一个凹向上质子库存表明，一个以上的质子在一个单一的过渡态转移，可能需要打开网站和释放产品的构象变化。反应的酸碱化学中涉及两个基团。这些基团之一催化谷氨酸的α-胺和AASA的醛之间形成亚胺的步骤。在V-1和V-1/K-Glu的pH-速率曲线中观察到pK（a）约为8的基团，必须质子化以获得最佳活性。在V-2和V-2/K-Sacc pH速率曲线中也观察到，需要未质子化。第二个基团的pK（a）为5.6，接受谷氨酸的α-胺的质子，因此它可以在AASA的羰基进攻时作为亲核试剂形成甲醇胺。
Saccharopine reductase (SR) [saccharopine dehydrogenase (L-glutamate forming), EC 1.5.1.10] catalyzes the condensation of L-alpha-aminoadipate-delta-semialdehyde (AASA) with L-glutamate to give an]mine, which is reduced by NADPH to give saccharopine. An acid-base chemical mechanism has been proposed for SR on the basis of pH-rate profiles and solvent deuterium kinetic isotope effects. A finite solvent isotope effect is observed indicating that proton(s) are in flight in the rate-limiting step(s) and likely the same step is limiting under both limiting and saturating substrate concentrations. A concave upward proton inventory suggests that more than one proton is transferred in a single transition state, likely a conformation change required to open the site and release products. Two groups are involved in the acid-base chemistry of the reaction. One of these groups catalyzes the steps involved in forming the imine between the alpha-amine of glutamate and the aldehyde of AASA. The group, which has a pK(a) of about 8, is observed in the pH-rate profiles for V-1 and V-1/K-Glu and must be protonated for optimal activity. It is also observed in the V-2 and V-2/K-Sacc pH-rate profiles and is required unprotonated. The second group, which has a pK(a) of 5.6, accepts a proton from the alpha-amine of glutamate so that it can act as a nucleophile in forming a carbinolamine upon attack of the carbonyl of AASA.