The putative catalytic bases have, at most, an accessory role in the mechanism of arginine kinase
The putative catalytic bases have, at most, an accessory role in the mechanism of arginine kinase
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DOI：
10.1074/jbc.m212931200
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发表时间：
2003-07-18
影响因子：
4.8
通讯作者：
Chapman, MS
中科院分区：
文献类型：
--
作者：
Pruett, PS;Azzi, A;Chapman, MS
精氨酸激酶是磷酸原激酶家族的一员，该家族包括肌酸激酶，并且可能在催化细胞 ATP 能量水平的缓冲方面具有共同的反应机制。通过催化碱从底物胍中提取质子长期以来被认为是早期的机械步骤。精氨酸激酶作为过渡态类似物复合物的结构（Zhou, G.、Somasundaram, T.、Blanc, E.、Parthasarathy, G.、Ellington, W. R. 和 Chapman, M. S. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 8449 - 8454）表明 Glu-225 和 Glu-314接触磷酸化氮的唯一潜在催化残基。在本研究中，在几种单位点和多位点突变酶中，这些残基被改变为天冬氨酸、谷氨酰胺和缬氨酸或丙氨酸。这些突变对底物结合常数影响很小。对活性的影响随 k(cat) 的降低而变化，从 3000 倍到小于 2 倍不等。一些突变体中显着活性的保留与已发表的同系物研究形成对比，表明这些残基的酸碱催化可能会提高速率，但不是绝对必要的。突变酶 E314D 在 1.9 埃分辨率下的晶体结构和 E225Q 在 2.8 埃分辨率下的晶体结构表明，底物的精确排列略有扭曲。因此，底物的预排序可能与酸碱化学、静电或这些残基对催化的适度影响的其他潜在影响一样重要。
Arginine kinase is a member of the phosphagen kinase family that includes creatine kinase and likely shares a common reaction mechanism in catalyzing the buffering of cellular ATP energy levels. Abstraction of a proton from the substrate guanidinium by a catalytic base has long been thought to be an early mechanistic step. The structure of arginine kinase as a transition state analog complex ( Zhou, G., Somasundaram, T., Blanc, E., Parthasarathy, G., Ellington, W. R., and Chapman, M. S. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 8449 - 8454) showed that Glu-225 and Glu-314 were the only potential catalytic residues contacting the phosphorylated nitrogen. In the present study, these residues were changed to Asp, Gln, and Val or Ala in several single and multisite mutant enzymes. These mutations had little impact on the substrate binding constants. The effect upon activity varied with reductions in k(cat) between 3000-fold and less than 2-fold. The retention of significant activity in some mutants contrasts with published studies of homologues and suggests that acid-base catalysis by these residues may enhance the rate but is not absolutely essential. Crystal structures of mutant enzymes E314D at 1.9 Angstrom and E225Q at 2.8 Angstrom resolution showed that the precise alignment of substrates is subtly distorted. Thus, pre-ordering of substrates might be just as important as acid-base chemistry, electrostatics, or other potential effects in the modest impact of these residues upon catalysis.