Reaction Mechanism of Alanine Racemase from Bacillus stearothermophilus
Reaction Mechanism of Alanine Racemase from Bacillus stearothermophilus
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DOI：
10.1074/jbc.m201615200
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发表时间：
2002-05
期刊：
影响因子：
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通讯作者：
A. Watanabe;T. Yoshimura;B. Mikami;H. Hayashi;H. Kagamiyama;N. Esaki
中科院分区：
文献类型：
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作者：
A. Watanabe;T. Yoshimura;B. Mikami;H. Hayashi;H. Kagamiyama;N. Esaki
在2-Å分辨率下测定了与反应中间体类似物N-(5‘-磷酸吡哆基)-L-丙氨酸(PLP-L-ALA)和N-(5’-磷酸吡哆基)-d-丙氨酸(PLP-d-ALA)结合的丙氨酸外消旋酶的晶体结构，PLP-L-ALA和PLP-d-ALA的结晶R因子分别为17.2和16.9。它们不仅彼此非常相似，而且与天然吡哆醛5‘-磷酸形式酶的结构也非常相似，三种结构在Cα的均方根偏差小于0.28Å。PLP-L-Ala和PLP-d-Ala周围氨基酸残基的侧链几乎相互重叠，与天然全酶PLP周围氨基酸残基的侧链几乎是重叠的。PLP-L-Ala的丙氨酸部分的α-H位于Tyr265‘的OH附近，而PLP-d-Ala的Lys-H位于Lys39的NZ附近。这支持了先前的发现，即Tyr265‘和Lys39分别是从L和d-丙氨酸中脱除α-氢的催化碱。这种双碱基机制的先决条件是从底物中提取的α-质子(直接或间接)在Lys39的NZ和Tyr265‘的OH之间转移，否则酶反应在一次转换后停止。只有PLP-Ala对映体中的羧基氧原子出现在一个合理的位置，氨基酸侧链和水分子都不在附近。因此，我们提出了丙氨酸外消旋酶的反应机理，底物羧基通过介导两个催化碱基Lys39和Tyr265‘之间的质子转移直接参与催化。分子轨道计算结果也支持这一机理。
The crystal structures of alanine racemase bound with reaction intermediate analogs,N-(5′-phosphopyridoxyl)-l-alanine (PLP-l-Ala) andN-(5′-phosphopyridoxyl)-d-alanine (PLP-d-Ala), were determined at 2.0-Å resolution with the crystallographic R factor of 17.2 for PLP-l-Ala and 16.9 for PLP-d-Ala complexes. They were quite similar not only to each other but also to the structure of the native pyridoxal 5′-phosphate (PLP)-form enzyme; root mean square deviations at Cα among the three structures were less than 0.28 Å. The side chains of the amino acid residues around the PLP-l-Ala and PLP-d-Ala were virtually superimposable on each other as well as on those around PLP of the native holoenzyme. The α-hydrogen of the alanine moiety of PLP-l-Ala was located near the OH of Tyr265′, whereas that of PLP-d-Ala was near the NZ of Lys39. These support the previous findings that Tyr265′ and Lys39 are the catalytic bases removing α-hydrogen from l- and d-alanine, respectively. The prerequisite for this two-base mechanism is that the α-proton abstracted from the substrate is transferred (directly or indirectly) between the NZ of Lys39 and the OH of Tyr265′; otherwise the enzyme reaction stops after a single turnover. Only the carboxylate oxygen atom of either PLP-Ala enantiomer occurred at a reasonable position that can mediate the proton transfer; neither the amino acid side chains nor the water molecules were located in the vicinity. Therefore, we propose a mechanism of alanine racemase reaction in which the substrate carboxyl group directly participates in the catalysis by mediating the proton transfer between the two catalytic bases, Lys39 and Tyr265′. The results of molecular orbital calculation also support this mechanism.