Avenier Frédéric, Domingos Josiel B, Vliet Liisa D Van, Hollfelder Florian
Contribution from the Department of Biochemistry, University of Cambridge, Cambridge CB2 1GA, United Kingdom.
J Am Chem Soc. 2007 Jun 20;129(24):7611-9. doi: 10.1021/ja069095g. Epub 2007 May 26.
The efficient integration of binding, catalysis, and multiple turnovers remains a challenge in building enzyme models. We report that systematic derivatization of polyethylene imine (PEI) with alkyl (C(2)-C(12)), benzyl, and guanidinium groups gives rise to catalysts ('synzymes') with rate accelerations (k(cat)/k(uncat)) of up to 10(4) for the intramolecular transesterification of 2-hydroxypropyl-p-nitrophenyl phosphate, HPNP, in the absence of metal. The synzymes exhibit saturation kinetics (K(M) approximately 250 microM, k(cat) approximately 0.5 min(-1)) and up to 2340 turnovers per polymer molecule. Catalysis can be specifically and competitively inhibited by anionic and hydrophobic small molecules. The efficacy of catalysis is determined by the PEI derivatization pattern. The derivatization reagents exert a synergistic effect, i.e., their combinations increase catalysis by more than the sum of each single modification. The pH-rate profile for k(cat)/K(M) is bell shaped with a maximum at pH 7.85 and can be explained as a combination of two effects that both have to be operative for optimal activity: K(M) increases at high pH due to deprotonation of PEI amines that bind the anionic substrate and kcat decreases as the availability of hydroxide decreases at low pH. Thus, catalysis is based on substrate binding by positively charged amine groups and the presence of hydroxide ion in active sites in an environment that is tuned for efficient catalysis. Inhibition studies suggest that the basis of catalysis and multiple turnovers is differential molecular recognition of the doubly negatively charged transition state (over singly charged ground state and product): this contributes a factor of at least 5-10-fold to catalysis and product release.
在构建酶模型时，将结合、催化及多次周转进行有效整合仍是一项挑战。我们报道，用烷基（C(2)-C(12)）、苄基和胍基对聚乙烯亚胺（PEI）进行系统衍生化，可得到催化剂（“模拟酶”），在无金属存在的情况下，对2-羟丙基-对硝基苯磷酸酯（HPNP）的分子内酯交换反应，其速率加速倍数（k(cat)/k(uncat)）高达10^4。这些模拟酶呈现饱和动力学（K(M)约为250微摩尔，k(cat)约为0.5分钟^(-1)），每个聚合物分子最多可周转2340次。催化作用可被阴离子和疏水性小分子特异性且竞争性地抑制。催化效果由PEI衍生化模式决定。衍生化试剂发挥协同作用，即它们的组合增强催化作用的幅度超过每种单一修饰增强作用之和。k(cat)/K(M)的pH-速率曲线呈钟形，在pH 7.85时达到最大值，这可解释为两种效应的组合，且这两种效应对于最佳活性均需起作用：在高pH时，由于结合阴离子底物的PEI胺去质子化，K(M)增大；在低pH时，随着氢氧根离子可用性降低，kcat减小。因此，催化作用基于带正电荷的胺基团对底物的结合以及在为高效催化而调节的环境中活性位点存在氢氧根离子。抑制研究表明，催化和多次周转的基础是对双负电荷过渡态（相对于单电荷基态和产物）的差异分子识别：这对催化和产物释放的贡献因子至少为5 - 10倍。
