Tawfik D S, Zemel R R, Arad-Yellin R, Green B S, Eshhar Z
Department of Pharmaceutical Chemistry, Faculty of Medicine, Hebrew University, Jerusalem, Israel.
Biochemistry. 1990 Oct 23;29(42):9916-21. doi: 10.1021/bi00494a023.
Monoclonal antibodies were raised against a mono-p-nitrophenyl phosphonate ester to elicit catalytic antibodies capable of hydrolyzing the analogous p-nitrophenyl ester or carbonate. Potential catalytic antibody producing clones were selected, by use of a competitive inhibition assay, on the basis of their affinity for a "short" transition-state analogue, a truncated hapten which maximizes the relative contribution of the transition-state structural elements to binding. Of 30-40 clones that would have been examined on the basis of hapten binding alone, 7 were selected and 4 of these catalyzed the hydrolysis of the relevant p-nitrophenyl ester. This competitive inhibition technique represents a general approach for selecting potential catalytic antibodies and significantly increases the probability of obtaining efficient catalytic monoclonal antibodies. Further study of the catalytic antibodies revealed significant rate enhancement (kcat/kuncat approximately 10(4)) and substrate specificity for the hydrolysis of the analogous ester and, for three of the antibodies, of the analogous carbonate. The antibodies displayed turnover, an essential feature of enzymes. Evidence that catalysis occurred at the antibody combining sites was provided by the identity of the binding and the catalysis-inhibition specificity patterns.
制备了针对单对硝基苯基膦酸酯的单克隆抗体，以产生能够水解类似对硝基苯基酯或碳酸酯的催化抗体。利用竞争性抑制试验，根据潜在的产生催化抗体的克隆对“短”过渡态类似物（一种截短的半抗原，其使过渡态结构元件对结合的相对贡献最大化）的亲和力，选择这些克隆。在仅基于半抗原结合而本来会被检测的30 - 40个克隆中，选择了7个，其中4个催化了相关对硝基苯基酯的水解。这种竞争性抑制技术代表了一种选择潜在催化抗体的通用方法，并显著提高了获得高效催化单克隆抗体的概率。对催化抗体的进一步研究揭示了显著的速率增强（kcat/kuncat约为10⁴）以及对类似酯水解的底物特异性，并且对于其中三种抗体，还显示出对类似碳酸酯水解的底物特异性。这些抗体表现出周转现象，这是酶的一个基本特征。结合特异性模式与催化 - 抑制特异性模式的一致性提供了催化发生在抗体结合位点的证据。
