Newby Zachary E R, Lau Edmond Y, Bruice Thomas C
Department of Chemistry and Biochemistry, University of California, Santa Barbara, CA 93106, USA.
Proc Natl Acad Sci U S A. 2002 Jun 11;99(12):7922-7. doi: 10.1073/pnas.122231499.
Ab initio and density functional calculations have been carried out to more fully understand the factors controlling the catalytic activity of the Thermus aquaticus DNA methyltransferase (MTaqI) in the N-methylation at the N(6) of an adenine nucleobase. The noncatalyzed reaction was modeled as a methyl transfer from trimethylsulfonium to the N(6) of adenine. Activation barriers of 32.0 kcal/mol and 24.0 kcal/mol were predicted for the noncatalyzed reaction in the gas phase by MP2/6-31+G(d,p)//HF/6-31+G(d,p) and B3LYP/6-31+G(d,p) calculations, respectively. Calculations performed to evaluate the effect of substrate positioning in the active site of MTaqI on the reaction determine the barrier to be 23.4 kcal/mol and 17.3 kcal/mol for the MP2/6-31+G(d,p)//HF/6-31+G(d,p) and B3LYP/6-31+G(d,p) gas phase calculations, respectively. The effect of hydrogen bonding between the N(6) of adenine and the terminal oxygen of Asn-105 on the activation barrier was also studied. A formamide molecule was modeled into the system to mimic the function of active site residue Asn-105. The activation barrier for this reaction was found to be 21.8 kcal/mol and 15.8 kcal/mol as determined from the MP2/6-31+G(d,p)//HF/6-31+G(d,p) and B3LYP/6-31+G(d,p) calculations, respectively. This result predicts a contribution of less than 2 kcal/mol to the lowering of the activation barrier from amide hydrogen bonding between formamide and N(6) of adenine. Comparison of the reaction coordinates suggest that it is not the hydrogen bonding of the Asn-105 that lends to the catalytic prowess of the enzyme since the organization of the substrates in the active site of the enzyme has a far greater effect on reducing the activation barrier. The results also suggest a stepwise mechanism for the removal of the hydrogen from the N(6) of adenine as opposed to a concerted reaction in which a proton is abstracted simultaneously with the transfer of the methyl group. The hydrogen on the N(6) of the intermediate methyl adenine product is far more acidic than in the reactant complex and may be subsequently abstracted by basic groups in the active site that are too weak to abstract the proton before the full sp(3) hybridization of the attacking nitrogen.
已进行从头算和密度泛函计算，以更全面地了解控制嗜热水栖菌DNA甲基转移酶（MTaqI）在腺嘌呤碱基N(6)处进行N-甲基化催化活性的因素。非催化反应被模拟为从三甲基锍到腺嘌呤N(6)的甲基转移。通过MP2/6-31+G(d,p)//HF/6-31+G(d,p)和B3LYP/6-31+G(d,p)计算，预测气相中非催化反应的活化能垒分别为32.0 kcal/mol和24.0 kcal/mol。为评估MTaqI活性位点中底物定位对反应的影响而进行的计算确定，对于MP2/6-31+G(d,p)//HF/6-31+G(d,p)和B3LYP/6-31+G(d,p)气相计算，活化能垒分别为23.4 kcal/mol和17.3 kcal/mol。还研究了腺嘌呤的N(6)与Asn-105的末端氧之间的氢键对活化能垒的影响。将一个甲酰胺分子引入系统以模拟活性位点残基Asn-105的功能。由MP2/6-31+G(d,p)//HF/6-31+G(d,p)和B3LYP/6-31+G(d,p)计算确定，该反应的活化能垒分别为21.8 kcal/mol和15.8 kcal/mol。该结果预测，甲酰胺与腺嘌呤N(6)之间的酰胺氢键对活化能垒降低的贡献小于2 kcal/mol。反应坐标的比较表明，并非Asn-105的氢键赋予了该酶催化能力，因为酶活性位点中底物的组织对降低活化能垒的影响要大得多。结果还表明，腺嘌呤N(6)上的氢的去除是一个逐步机制，而不是在甲基转移的同时质子被夺取的协同反应。中间甲基腺嘌呤产物N(6)上的氢比反应物复合物中的氢酸性强得多，并且可能随后被活性位点中的碱性基团夺取，这些碱性基团在进攻氮完全进行sp(3)杂化之前太弱而无法夺取质子。
