Abstract
A monoclonal antibody (MAb) was produced against thep-nitrophenylphosphate derivative of 3α,5β-lithocholic acid, a transition-state analog for hydrolysis of a steroidalp-nitrophenylcarbonate. The indicated reaction was catalyzed by this Ab with kinetic constants kcat = 4.0 × 10-2min and Km = 3.3 μM at pH 9.0 and 35°C. The Ab also hydrolyzed the isomericp-nitrophenylcarbonate of 3β,5β-lithocholic acid with kcat = 8.4 × 10-2/min and Km = 1.0 μM. Bovine serum albumin (BSA) was found to catalyze the same reactions with similar turnover rates and Michaelis constants of 15 and 14 μM, respectively. Although the BSA-catalyzed reaction was only weakly inhibited by the phosphate ester TSA (IC50 ca. 40 μM), the Ab-catalyzed reaction was completely inhibited at less than 1 μM of the TSA. The relative rates and efficiencies of the MAbcatalyzed and BSA-catalyzed reactions are discussed in the context of the hydrophobic sites and intrinsic reactivity of the protein surfaces, and the induction of groups on the Ab to enhance the enzymatic function.
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Riva, S., Mendozza, M., Carrea, G. et al. Comparison of antibody and albumin catalyzed hydrolysis of steroidalp-nitrophenylcarbonates. Appl Biochem Biotechnol 75, 33–44 (1998). https://doi.org/10.1007/BF02787707
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DOI: https://doi.org/10.1007/BF02787707