Baldwin E, Schultz P G
Department of Chemistry, University of California, Berkeley 94720.
Science. 1989 Sep 8;245(4922):1104-7. doi: 10.1126/science.2672338.
A hybrid Fv fragment of the dinitrophenyl-binding immunoglobulin A (IgA), MPOC315, has been generated by reconstituting a recombinant variable light chain (VL) produced in Escherichia coli with a variable heavy chain (VH) derived from the antibody. The Tyr34 residue of VL was substituted by His in order to introduce a catalytic imidazole into the combining site for the ester hydrolysis. The His mutant Fv accelerated the hydrolysis of the 7-hydroxycoumarin ester of 5-(2,4-dinitrophenyl)-aminopentanoic acid 90,000-fold compared to the reaction with 4-methyl imidazole at pH 6.8 and had an initial rate that was 45 times as great as that for the wild-type Fv. The hydrolyses of aminopropanoic and aminohexanoic homologs were not significantly accelerated. Thus a single deliberate amino acid change can introduce significant catalytic activity into an antibody-combining site, and chemical modification data can be used to locate potential sites for the introduction of catalytic residues.
通过将在大肠杆菌中产生的重组轻链可变区（VL）与源自该抗体的重链可变区（VH）重组，生成了二硝基苯基结合免疫球蛋白A（IgA）的杂合Fv片段MPOC315。为了将催化性咪唑引入酯水解的结合位点，VL的Tyr34残基被His取代。与在pH 6.8下与4-甲基咪唑的反应相比，His突变体Fv使5-（2,4-二硝基苯基）-氨基戊酸的7-羟基香豆素酯的水解加速了90,000倍，其初始速率是野生型Fv的45倍。氨基丙酸和氨基己酸同系物的水解没有明显加速。因此，单一的有意氨基酸变化可以将显著的催化活性引入抗体结合位点，并且化学修饰数据可用于定位引入催化残基的潜在位点。
