Potter: Structures of carboxylesterases

Crystal structure of the rabbit liver carboxylesterase

Alternative strategies to improve the antitumor efficacy have concentrated upon the design of novel camptothecin analogs. To effect this, we have determined the x-ray crystal structures of the rabbit liver carboxylesterase and hCE1 (see figure below). These studies, performed in collaboration with Dr. Matthew Redinbo (University of North Carolina), have indicated that the active site of these enzymes is buried at the end of a deep gorge in the protein. Additionally, we have identified a ‘side door’ in the molecule that may allow the release of the acid fragment generated by hydrolysis of the ester. Current experiments are aimed at determining the role of the ‘side door’ in carboxylesterase-mediated catalysis. In addition, we are synthesizing novel CPT-11 analogs that would be expected to be activated more efficiently by human CEs, and hence may demonstrate greater antitumor activity.

In addition, we have determined the crystal structures of a human liver carboxylesterase, hCE1. This enzyme is greater than 80% homologous to the rabbit liver carboxylesterase but cannot activate CPT-11. Structural studies indicate that this is due to differences in the amino acids that form the entrances to the active site entrances of the proteins. In the rabbit protein, these domains are highly flexible and hence this enzyme can accommodate larger, more bulky substrates within the active site. In hCE1, these loops are rigid and therefore accessibility of molecules to the catalytic amino acids buried at the bottom of the gorge is dictated by their size and shape. We have recently generated a variant form of hCE1 containing selected residues present in the rabbit enzyme. This protein termed hCE1m6 is as efficient at activating CPT-11 as the lagomorph enzyme and is currently being used for enzyme-prodrug strategies. Furthermore, we are determining the structure of hCE1m6 with the goal of using this information to enhance prodrug activation.