Mode of binding of methyl acceptor substrates to the adrenaline-synthesizing enzyme phenylethanolamine N-methyltransferase:: Implications for catalysis
Mode of binding of methyl acceptor substrates to the adrenaline-synthesizing enzyme phenylethanolamine N-methyltransferase:: Implications for catalysis
复制标题
DOI：
10.1021/bi051636b
复制
发表时间：
2005-12-27
期刊：
影响因子：
2.9
通讯作者：
Martin, JL
中科院分区：
文献类型：
--
作者：
Gee, CL;Tyndall, JDA;Martin, JL
在这里，我们报道了三种人类苯乙醇胺n -甲基转移酶（PNMT）的晶体结构配合物，其中一种与含有柔性乙醇胺侧链（poctopamine）的底物结合，另一种与半刚性类似底物结合[顺式-(1R,2S)-2-氨基-1-四醇，顺式-(1R,2S)- at]，第三种与反式-(1S,2S)-2-氨基-1-四醇[反式-(1S,2S)- at]结合，后者作为PNMT的抑制剂而不是底物。对章鱼胺的柔性乙醇胺基团的关键β -羟基与酸性残基Asp267之间的水介导相互作用可能在正确定位侧链以使胺发生甲基化方面发挥关键作用。与Glu219的第二次相互作用可能起的作用较小。催化作用可能是通过Glu185作用下胺的去质子化发生的；该残基的突变显著降低了k(cat)，但不影响k -m。顺式-(1R,2S)- at的结合模式支持了这种底物是柔性PNMT底物构象受限的类似物的观点，因为它与对章鱼胺观察到的酶形成了类似的相互作用。相比之下，陷阱-(1S,2S)- at是一种抑制剂，而不是底物，与顺式-(1R,2S)- at相比，其结合方向翻转了180度。这种翻转结合模式的结果是羟基与Asp267和Glu219之间的相互作用丢失。然而，抑制剂陷阱的胺-(1S,2S)- at和底物顺式-(1R,2S)- at都在辅因子的甲基转移距离内。这些结果表明，PNMT只有在“锚定”相互作用（如对章鱼胺的β羟基和顺式at）存在时，才能催化甲基向配体胺的转移。
Here we report three crystal structure complexes of human phenylethanolamine N-methyl-transferase (PNMT), one bound with a substrate that incorporates a flexible ethanolamine side chain (poctopamine), a second bound with a semirigid analogue substrate [cis-(1R,2S)-2-amino-1-tetralol, cis-(1R,2S)-AT], and a third with trans-(1S,2S)-2-amino-1-tetralol [trans-(1S,2S)-AT] that acts as an inhibitor of PNMT rather than a substrate. A water-mediated interaction between the critical beta-hydroxyl of the flexible ethanolamine group of p-octopamine and an acidic residue, Asp267, is likely to play a key role in positioning the side chain correctly for methylation to occur at the amine. A second interaction with Glu219 may play a lesser role. Catalysis likely occurs via deprotonation of the amine through the action of Glu185; mutation of this residue significantly reduced the k(cat) without affecting the K-m. The mode of binding of cis-(1R,2S)-AT supports the notion that this substrate is a conformationally restrained analogue of flexible PNMT substrates, in that it forms interactions with the enzyme similar to those observed for p-octopamine. By contrast, traps-(1S,2S)-AT, an inhibitor rather than a substrate, binds in an orientation that is flipped by 180 degrees compared with cis-(1R,2S)-AT. A consequence of this flipped binding mode is that the interactions between the hydroxyl and Asp267 and Glu219 are lost. However, the amines of inhibitor traps-(1S,2S)-AT and substrate cis-(1R,2S)-AT are both within methyl transfer distance of the cofactor. These results suggest that PNMT catalyzes transfer of methyl to ligand amines only when "anchor" interactions, such as those identified for the beta-hydroxyls of p-octopamine and cis-AT, are present.