Biocatalysis of amide and peptide bond synthesis by cocaine esterase and α-amino acid ester hydrolase

Abstract

Amide and peptide bonds-containing compounds are ubiquitous in Nature and popular in the pharmaceutical and chemical industries. Amide bond synthesis occurs with an un-activated (thermodynamic control: carboxylic acid) or activated (kinetic control: carboxylic ester) and an amine an as the starting materials. With the goal to develop an affordable, readily available, and substrate independent assay, a pH-based assay was conceived with phenol red as the dye of choice for studies. In addition to the assay, using the pKa’s of substrates, products, and buffers as well as the absorbance of dye, a mathematical model was developed to calculate pH and predict, conversion of substrate into the products. The assay was capable of distinguishing synthesis of amide from hydrolysis of ester. Using the assay, substrate and reaction promiscuity of cocaine esterase was explored. Cocaine esterase (CocE) was found to have distinctive properties compared to α-amino acid ester hydrolase (AEH). While AEH is capable of synthesizing β-lactam antibiotics such as ampicillin, CocE is not. While CocE is able to take one of the starting materials of ampicillin synthesis, phenylglycine methyl ester, to synthesize dipeptides, AEH shows no such activity. The preliminary results show that CocE has greater preference for amide synthesis over that of amide hydrolysis. Finally, while AEH only accepts α-amino compounds, CocE has the tendency to polymerize such compounds if they are esters; CocE is also capable of accepting various non-amino acid esters with notable chemical features. CocE prefers aromatic compounds to those with alkyl moieties as leaving groups or acyl moieties. The opposite was found to be true with cases in which the acyl moiety is a β-keto compound. The results add to the concept of enzyme-structure-activity relationship for CocE. More importantly, it begins to lay the foundation from which amide synthesis activity of CocE can be engineered.