中科院大連化學(xué)物理研究所復(fù)雜分子體系反應(yīng)動(dòng)力學(xué)研究組（1101組）與該所高級(jí)伙伴研究員,、美國(guó)肯塔基大學(xué)湛昌國(guó)教授合作，在細(xì)胞色素P450 2A6催化尼古丁反應(yīng)機(jī)理研究方面取得了新進(jìn)展,。
酶催化反應(yīng)機(jī)理是當(dāng)今計(jì)算化學(xué)研究中的前沿領(lǐng)域和熱門課題,。理論計(jì)算作為一種有力的手段，能夠研究酶催化反應(yīng)的過(guò)程,，解釋實(shí)驗(yàn)現(xiàn)象,，闡明反應(yīng)機(jī)理，并為進(jìn)一步的實(shí)驗(yàn)研究提供理論基礎(chǔ)。尼古丁是煙草中的主要成癮物質(zhì),，它在人體中的代謝主要是由細(xì)胞色素P450 2A6（CYP2A6）催化的,，研究尼古丁和CYP2A6的結(jié)合方式以及CYP2A6催化尼古丁的反應(yīng)機(jī)理能夠?yàn)楹侠碓O(shè)計(jì)CYP2A6抑制劑提供理論支持,，為戒煙藥物的研發(fā)提供一定的理論基礎(chǔ)。
細(xì)胞色素P450 2A6（CYP2A6）
大連化物所1101組自2006年以來(lái)一直致力于酶催化反應(yīng)機(jī)理的理論研究,，并相繼在J. Phys. Chem. B; ChemBioChem; J. Biol. Inorg. Chem.; Dalton Trans.等雜志上發(fā)表了一系列文章,。近期，韓克利研究員等人與湛昌國(guó)教授合作,，利用分子對(duì)接,、分子動(dòng)力學(xué)模擬、QM/MM計(jì)算等方法,，研究了尼古丁與CYP2A6的結(jié)合方式、CYP2A6催化的尼古丁5'-羥基化反應(yīng)的機(jī)理以及5'-羥基化反應(yīng)的立體選擇性,。結(jié)果表明,，雖然尼古丁在溶液中主要以質(zhì)子化的形式存在，但是它以非質(zhì)子化的形式(SRt和SRc)結(jié)合到CYP2A6的活性中心,。在CYP2A6-尼古丁復(fù)合物中,，尼古丁吡啶環(huán)的N與CYP2A6的Asn297形成氫鍵,，吡啶環(huán)與周圍的苯丙氨酸存在π-π相互作用,，吡咯烷環(huán)的trans-5'-H或者cis-5'-H指向CYP2A6的活性中心Cpd I的O,，從而利于5'-羥基化反應(yīng)的發(fā)生。
研究發(fā)現(xiàn),，CYP2A6催化的尼古丁5'-羥基化反應(yīng)由兩步組成：第一步是尼古丁的5'-H傳遞到Cpd I的O上（氫傳遞）,；第二步是OH再與尼古丁的5'-位置的C結(jié)合，形成5'-羥基尼古?。ㄑ醴磸棧?。其中，氫傳遞是決速步驟,。計(jì)算結(jié)果顯示,，CYP2A6催化的尼古丁5'-羥基化反應(yīng)容易發(fā)生在trans-5'-H上,，反應(yīng)的立體選擇性為97%,，與實(shí)驗(yàn)報(bào)導(dǎo)的立體選擇性相符。（生物谷Bioon.com）
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American Chemical Society DOI: 10.1021/ja111657j
Catalytic Mechanism of Cytochrome P450 for 5′-Hydroxylation of Nicotine: Fundamental Reaction Pathways and Stereoselectivity
Dongmei Li, Xiaoqin Huang, Keli Han, and Chang-Guo Zhan
A series of computational methods were used to study how cytochrome P450 2A6 (CYP2A6) interacts with (S)-(-)-nicotine, demonstrating that the dominant molecular species of (S)-(-)-nicotine in CYP2A6 active site exists in the free base state (with two conformations, SRt and SRc), despite the fact that the protonated state is dominant for the free ligand in solution. The computational results reveal that the dominant pathway of nicotine metabolism in CYP2A6 is through nicotine free base oxidation. Further, first-principles quantum mechanical/molecular mechanical free energy (QM/MM-FE) calculations were carried out to uncover the detailed reaction pathways for the CYP2A6-catalyzed nicotine 5′-hydroxylation reaction. In the determined CYP2A6–(S)-(-)-nicotine binding structures, the oxygen of Compound I (Cpd I) can abstract a hydrogen from either the trans-5′- or the cis-5′-position of (S)-(-)-nicotine. CYP2A6-catalyzed (S)-(-)-nicotine 5′-hydroxylation consists of two reaction steps, that is, the hydrogen transfer from the 5′-position of (S)-(-)-nicotine to the oxygen of Cpd I (the H-transfer step), followed by the recombination of the (S)-(-)-nicotine moiety with the iron-bound hydroxyl group to generate the 5′-hydroxynicotine product (the O-rebound step). The H-transfer step is rate-determining. The 5′-hydroxylation proceeds mainly with the stereoselective loss of the trans-5′-hydrogen, that is, the 5′-hydrogen trans to the pyridine ring. The calculated overall stereoselectivity of 97% favoring the trans-5′-hydroxylation is close to the observed stereoselectivity of 89–94%. This is the first time it has been demonstrated that a CYP substrate exists dominantly in one protonation state (cationic species) in solution, but uses its less-favorable protonation state (neutral free base) to perform the enzymatic reaction.
