Probing the chemical mechanism of saccharopine reductase from Saccharomyces cerevisiae using site-directed mutagenesis.
Probing the chemical mechanism of saccharopine reductase from Saccharomyces cerevisiae using site-directed mutagenesis.
复制标题
利用定点诱变探讨酿酒酵母糖碱还原酶的化学机制。
DOI：
10.1016/j.abb.2015.08.023
复制
发表时间：
2015
影响因子：
3.9
通讯作者：
Cook,PaulF
中科院分区：
文献类型：
--
作者：
Vashishtha,AshwaniK;West,AnnH;Cook,PaulF
糖胺还原酶催化l-α-氨基己二酸酯-δ-半醛与l-谷氨酸的还原氨化反应生成糖胺。已经提出了该还原酶的两种机制，一种是利用酶侧链作为酸碱催化基团，另一种是由酶结合的反应物催化反应。利用定点突变技术改变还原酶活性部位的酸碱候选者，消除其可电离侧链。从而获得了D126A、C154S、Y99F和几种双突变酶。谷氨酸形成方向的动力学参数略有下降，与酸碱催化剂的损失不一致。所有突变酶的pH-速率曲线在低pH和高pH下都降低，表明所有酶中仍然存在酸和碱催化基团。溶剂动力学的氚同位素效应都大于野生型酶，且彼此大致相同，表明慢步骤与野生型酶相同，即开放位点和释放产物的构象变化(沿糖胺形成的方向)。总体而言，酸碱化学很可能是由结合的反应物催化的，除了谷氨酸的α-胺的去质子化，这可能需要酶残基。
Saccharopine reductase catalyzes the reductive amination ofl-α-aminoadipate-δ-semialdehyde withl-glutamate to give saccharopine. Two mechanisms have been proposed for the reductase, one that makes use of enzyme side chains as acid–base catalytic groups, and a second, in which the reaction is catalyzed by enzyme-bound reactants. Site-directed mutagenesis was used to change acid–base candidates in the active site of the reductase to eliminate their ionizable side chain. Thus, the D126A, C154S and Y99F and several double mutant enzymes were prepared. Kinetic parameters in the direction of glutamate formation exhibited modest decreases, inconsistent with the loss of an acid–base catalyst. The pH-rate profiles obtained with all mutant enzymes decrease at low and high pH, suggesting acid and base catalytic groups are still present in all enzymes. Solvent kinetic deuterium isotope effects are all larger than those observed for wild type enzyme, and approximately equal to one another, suggesting the slow step is the same as that of wild type enzyme, a conformational change to open the site and release products (in the direction of saccharopine formation). Overall, the acid–base chemistry is likely catalyzed by bound reactants, with the exception of deprotonation of the α-amine of glutamate, which likely requires an enzyme residue.
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[157] 赖氨酸生物合成（酵母）
DOI：
--
发表时间：
1971
期刊：
影响因子：
--
作者：
H. P. Broquist
通讯作者：
H. P. Broquist
影响因子：
7
作者：
Nishida, H;Nishiyama, M;Yamane, H
通讯作者：
Yamane, H
酿酒酵母糖碱脱氢酶（L-谷氨酸形成）的总体动力学机制。
影响因子：
2.9
作者：
Vashishtha,AshwaniKumar;West,AnnH;Cook,PaulF
通讯作者：
Cook,PaulF
影响因子：
2.9
作者：
Vashishtha, Ashwani Kumar;West, Ann H.;Cook, Paul F.
通讯作者：
Cook, Paul F.
DOI：
10.4324/9780203833575
发表时间：
2007-03
期刊：
--
影响因子：
--
作者：
Paul F. Cook;Paul F. Cook;W. W. Cleland-W.
通讯作者：
Paul F. Cook;Paul F. Cook;W. W. Cleland-W.