Dobson Renwick C J, Devenish Sean R A, Turner Leighton A, Clifford Veronica R, Pearce F Grant, Jameson Geoffrey B, Gerrard Juliet A
School of Biological Sciences, University of Canterbury, Private Bag 4800, Christchurch, New Zealand.
Biochemistry. 2005 Oct 4;44(39):13007-13. doi: 10.1021/bi051281w.
In plants and bacteria, the branch point of (S)-lysine biosynthesis is the condensation of (S)-aspartate-beta-semialdehyde [(S)-ASA] and pyruvate, a reaction catalyzed by dihydrodipicolinate synthase (DHDPS, EC 4.2.1.52). It has been proposed that Arg138, a residue situated at the entrance to the active site of DHDPS, is responsible for binding the carboxyl of (S)-ASA and may additionally be involved in the mechanism of (S)-lysine inhibition. This study tests these assertions by mutation of Arg138 to both histidine and alanine. Following purification, DHDPS-R138H and DHDPS-R138A each showed severely compromised activity (approximately 0.1% that of the wild type), and the apparent Michaelis-Menten constant for (S)-ASA in each mutant, calculated using a pseudo-single substrate analysis, was significantly higher than that of the wild type. This provides good evidence that Arg138 is indeed essential for catalysis and plays a key role in substrate binding. To test whether structural changes could account for the change in kinetic behavior, the solution structure was probed via far-UV circular dichroism, confirming that the mutations at position 138 did not modify secondary structure. The crystal structures of both mutant enzymes were determined, confirming the presence of the mutations and suggesting that Arg138 plays an important role in catalysis: the stabilization of the catalytic triad residues, a motif we have previously demonstrated to be essential for activity. In addition, the role of Arg138 in (S)-lysine inhibition was examined. Both mutant enzymes showed the same IC(50) values as the wild type but different partial inhibition patterns, from which it is concluded that arginine 138 is not essential for (S)-lysine inhibition.
在植物和细菌中，（S）-赖氨酸生物合成的分支点是（S）-天冬氨酸-β-半醛[（S）-ASA]与丙酮酸的缩合反应，该反应由二氢吡啶二羧酸合酶（DHDPS，EC 4.2.1.52）催化。有人提出，位于DHDPS活性位点入口处的残基Arg138负责结合（S）-ASA的羧基，并且可能还参与了（S）-赖氨酸抑制机制。本研究通过将Arg138分别突变为组氨酸和丙氨酸来验证这些说法。纯化后，DHDPS-R138H和DHDPS-R138A的活性均严重受损（约为野生型的0.1%），使用伪单底物分析计算得出的每个突变体中（S）-ASA的表观米氏常数均显著高于野生型。这充分证明Arg138确实是催化作用所必需的，并且在底物结合中起关键作用。为了测试结构变化是否可以解释动力学行为的变化，通过远紫外圆二色性探测溶液结构，证实138位的突变并未改变二级结构。测定了两种突变酶的晶体结构，证实了突变的存在，并表明Arg138在催化中起重要作用：稳定催化三联体残基，我们之前已证明该基序对活性至关重要。此外，还研究了Arg138在（S）-赖氨酸抑制中的作用。两种突变酶的IC50值与野生型相同，但部分抑制模式不同，由此得出结论，精氨酸138对（S）-赖氨酸抑制并非必需。
