Lindner Ariel B, Kim Se Ho, Schindler Dan G, Eshhar Zelig, Tawfik Dan S
Department of Immunology, Weizmann Institute of Science, Rehovot 76100, Israel.
J Mol Biol. 2002 Jul 12;320(3):559-72. doi: 10.1016/s0022-2836(02)00418-7.
Understanding enzymes quantitatively and mimicking their remarkable catalytic efficiency is a paramount challenge. Here, we applied esterolytic antibodies (the D-Abs) to dissect and quantify individual elements of enzymatic catalysis such as transition state (TS) stabilization, nucleophilic reactivity and conformational changes. Kinetic and mutagenic analysis of the D-Abs were combined with existing structural evidence to show that catalysis by the D-Abs is driven primarily by stabilization of the tetrahedral oxyanionic intermediate of ester hydrolysis formed by the nucleophilic attack of an exogenous (solution) hydroxide anion. The side-chain of TyrH100d is shown to be the main H-bond donor of the D-Abs oxyanion hole. The pH-rate and pH-binding profiles indicate that the strength of this H-bond increases dramatically as the neutral substrate develops into the oxyanionic TS, resulting in TS stabilization of 5-7 kcal/mol, which is comparable to oxyanionic TS stabilization in serine hydrolases. We show that the rate of the exogenous (intermolecular) nucleophilic attack can be enhanced by 2000-fold by replacing the hydroxide nucleophile with peroxide, an alpha-nucleophile that is much more reactive than hydroxide. In the presence of peroxide, the rate saturates (k(cat)(max)) at 6 s(-1). This rate-ceiling appears to be dictated by the rate of the induced-fit conformational rearrangement leading to the active antibody-TS complex. The selective usage of negatively charged exogenous nucleophiles by the D-Abs led to the identification of a positively charged channel. Imprinted by the negatively-charged TS-analogue against which these antibodies were elicited, this channel presumably directs the nucleophile to the antibody-bound substrate. Our findings are discussed in comparison with serine esterases and, in particular, with cocaine esterase (cocE), which possesses a tyrosine based oxyanion hole.
定量理解酶并模拟其卓越的催化效率是一项至关重要的挑战。在此，我们应用酯解抗体（D-Abs）来剖析和量化酶催化的各个要素，如过渡态（TS）稳定、亲核反应性和构象变化。将D-Abs的动力学和诱变分析与现有的结构证据相结合，结果表明D-Abs的催化主要由外源（溶液中的）氢氧根阴离子亲核攻击形成的酯水解四面体氧阴离子中间体的稳定所驱动。TyrH100d的侧链被证明是D-Abs氧阴离子空穴的主要氢键供体。pH-速率和pH-结合曲线表明，随着中性底物发展为氧阴离子TS，该氢键的强度急剧增加，导致TS稳定化5-7千卡/摩尔，这与丝氨酸水解酶中的氧阴离子TS稳定化相当。我们表明，通过用过氧化物替代氢氧根亲核试剂，外源（分子间）亲核攻击的速率可提高2000倍，过氧化物是一种比氢氧根更具反应性的α-亲核试剂。在过氧化物存在下，速率在6 s⁻¹时达到饱和（k(cat)(max)）。这个速率上限似乎由诱导契合构象重排形成活性抗体-TS复合物的速率所决定。D-Abs对带负电荷的外源亲核试剂的选择性使用导致了一个带正电荷通道的识别。该通道由引发这些抗体的带负电荷的TS类似物印记，大概将亲核试剂导向抗体结合的底物。我们将研究结果与丝氨酸酯酶，特别是与具有基于酪氨酸的氧阴离子空穴的可卡因酯酶（cocE）进行了比较讨论。
