Conformational change in aspartate aminotransferase on substrate binding induces strain in the catalytic group and enhances catalysis
Conformational change in aspartate aminotransferase on substrate binding induces strain in the catalytic group and enhances catalysis
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DOI：
10.1074/jbc.m209235200
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发表时间：
2003-03-14
影响因子：
4.8
通讯作者：
Kagmiyama, H
中科院分区：
文献类型：
--
作者：
Hayashi, H;Mizuguchi, H;Kagmiyama, H
已知天冬氨酸氨基转移酶在底物结合时会发生显著的构象变化，其中小结构域接近大结构域，活性部位入口处的残基聚集在一起。伴随着这种构象变化的是吡哆醛5‘-磷酸-赖氨酸(258)乙二胺的pK(A)增加了两个单位，这被认为是增强催化的。为了阐明构象变化是如何与乙二胺PKA的位移相耦合的，以及这些变化是如何参与催化的，我们从结构和动力学上分析了一种酶，其中位于结构域界面和活性中心入口处的Val(39)被更大的残基Phe取代。V39F突变体酶的构象更加开放，乙二胺pK(A)比野生型酶降低了0.7个单位。当Asn(194)预先被Ala取代时，V39F突变并没有减少乙二胺PK(A)，表明结构域旋转通过Arg(386)-Asn(194)-吡哆醛5‘-磷酸连接系统控制乙二胺PK(A)。顺丁烯二酸结合的V39F酶的乙二胺PK(A)比顺丁烯二酸结合的野生型酶低0.9单位。然而，马来酸、Asn(194)和Arg(386)的位置在突变体酶和野生型酶之间是重叠的；因此，结构域旋转不是乙二胺pK(A)值降低的原因。顺丁烯二酸结合的V39F酶在37-39区的侧链堆积模式发生了变化，Gly(38)羰基O与Tyr(225)Oeta之间缺乏排斥力似乎是pK(A)值降低的原因。动力学分析表明，排斥力提高了米氏络合物的自由能，促进了催化反应。
Aspartate aminotransferase has been known to undergo a significant conformational change, in which the small domain approaches the large domain, and the residues at the entrance of the active site pack together, on binding of substrates. Accompanying this conformational change is a two-unit increase in the pK(a) of the pyridoxal 5'-phosphate-Lys(258) aldimine, which has been proposed to enhance catalysis. To elucidate how the conformational change is coupled to the shift in the aldimine pKa and how these changes are involved in catalysis, we analyzed structurally and kinetically an enzyme in which Val(39) located at both the domain interface and the entrance of the active site was replaced with a bulkier residue, Phe. The V39F mutant enzyme showed a more open conformation, and the aldimine pK(a) was lowered by 0.7 unit compared with the wild-type enzyme. When Asn(194) had been replaced by Ala in advance, the V39F mutation did not decrease the aldimine pK(a), showing that the domain rotation controls the aldimine pK(a) via the Arg(386)-Asn(194)-pyridoxal 5'-phosphate linkage system. The maleate-bound V39F enzyme showed the aldimine pK(a) 0.9 unit lower than that of the maleate-bound wild-type enzyme. However, the positions of maleate, Asn(194), and Arg(386) were superimposable between the mutant and the wild-type enzymes; therefore, the domain rotation was not the cause of the lowered aldimine pK(a) value. The maleate-bound V39F enzyme showed an altered side-chain packing pattern in the 37-39 region, and the lack of repulsion between Gly(38) carbonyl O and Tyr(225) Oeta seemed to be the cause of the reduced pK(a) value. Kinetic analysis suggested that the repulsion increases the free energy level of the Michaelis complex and promotes the catalytic reaction.