中文摘要：

研究了严重急性呼吸系统综合症（SARS）冠状病毒3C-Like蛋白酶（3CLpro）在存在底物或抑制剂时的二聚体形成情况.通过测定酶活性随酶浓度的变化,拟合出在底物存在下酶二聚体的解离常数约为0.94μmol·L-1,小于纯蛋白酶的二聚体解离常数（14.0μmol·L-1）,表明底物对二聚体的形成具有增强作用.选用与底物具有类似结合方式的靛红类抑制剂N-萘甲基靛红-5-甲酰胺（5f）,利用超速离心沉降速率方法定量测定了SARS 3CL蛋白酶单体和二聚体在不同浓度5f时的含量,发现5f同样具有诱导二聚体形成的能力.在3μmol·L-1蛋白酶浓度下测定得到诱导二聚的EC50值（半数有效浓度）约为1μmol·L-1,说明二聚体中只有一个单体与抑制剂结合.研究结果表明,随着底物浓度的升高,SARS 3CL蛋白酶会形成更多的二聚体,而二聚体含量的提高又反过来提高酶的活性,这种双向别构调控机制有可能是病毒用来调控多聚蛋白水解速率和组装时机的一种方法.

英文摘要：

The 3C-like proteinase （3CLpro） of severe acute respiratory syndrome （SARS） coronavirus has been proposed to be a key target for anti-SARS drug discovery.It has been proposed and verified that the dimer was the active form of 3CLpro and only one protomer is active.In our previous work,we measured the dissociation constant （Kd） of the purified SARS 3CLpro using analytical ultracentrifugation at around 14.0 μmol·L-1.Using this Kd value,most of the SARS 3CLpro in the in vitro activity assay （1-3 μmol·L-1） might be in the monomer form and inactive.To explain this dilemma,we measured the enzyme activity change together with the enzyme concentration.By fitting the concentration dependent activity profile,the apparent dissociation constant was found to be 0.94 μmol·L-1,indicating a clear tendency toward substrate enhanced dimerization.This also explains why SARS 3CLpro was still active in the in vitro activity assay under a relatively low enzyme concentration.To further verify the substrate induced dimerization phenomenon,we selected a previously reported SARS 3CLpro isatin inhibitor,1-（2-naphthlmethyl） isatin-5-carboxamide （5f）,which has similar binding interactions with the substrate and we studied its influence on SARS 3CLpro dimer formation using analytical ultracentrifugation.5f showed a strong ability to induce SARS 3CLpro dimer formation.By measuring the dimer and monomer distribution under different 5f concentrations,the EC50 of dimer induction was found to be about 1.0 μmol·L-1 under an enzyme concentration of 3.0 μmol· L-1.This implies that only one protomer in the SARS 3CLpro dimer binds to the inhibitor or the substrate.As the apparent association constant and thus the enzyme activity of SARS 3CLpro increases with the concentration of the substrate,this may be a smart way to allosterically regulate the hydrolysis of the SARS viral polyproteins and the correct assembly of virons.