Aldolase antibodies that operate via an enamine mechanism were developed by in vitro selection. Antibody Fab phage display libraries were created where the catalytic active site residues of aldolase antibodies 38C2 and 33F12 were combined with a naive human antibody V gene repertoire. Selection from these libraries with 1,3-diketones covalently trapped the amino groups of reactive lysine residues by formation of stable enaminones. The selected aldolase antibodies retained the essential catalytic lysine residue and its function in altered and humanized primary antibody structures. The substrate specificity of the aldolase antibodies was directly related to the structure of the diketone used for selection. The kcat values of the antibody-catalyzed retro-aldol reactions were correlated with the Kd values, i.e. the reactivities of the selected aldolase antibodies for the corresponding diketones. Antibodies that bound to the diketone with a lower Kd value displayed a higher kcat value in the retro-aldol reaction, and a linear relationship was observed in the plots of logkcat versus logKd. These results indicate that selections with diketones directed the evolution of aldolase antibodies in vitro that operate via an enamine mechanism. This strategy provides a route to tailor-made aldol catalysts with different substrate specificities.
|Number of pages||12|
|Journal||Journal of Molecular Biology|
|State||Published - 23 Jan 2004|
- Aldolase antibody
- Catalytic antibody
- In vitro evolution
- Phage display