title: Comparison of antibody and albumin catalyzed hydrolysis of steroidal p-nitrophenylcarbonates.
abstract: A monoclonal antibody (MAb) was produced against the p-nitrophenylphosphate derivative of 3 alpha, 5 beta-lithocholic acid, a transition-state analog for hydrolysis of a steroidal p-nitrophenylcarbonate. The indicated reaction was catalyzed by this Ab with kinetic constants kcat = 4.0 x 10(-2)/min and K(m) = 3.3 microM at pH 9.0 and 35 degrees C. The Ab also hydrolyzed the isomeric p-nitrophenylcarbonate of 3 beta, 5 beta-lithocholic acid with kcat = 8.4 x 10(-2)/min and K(m) = 1.0 microM. Bovine serum albumin (BSA) was found to catalyze the same reactions with similar turnover rates and Michaelis constants of 15 and 14 microM, respectively. Although the BSA-catalyzed reaction was only weakly inhibited by the phosphate ester TSA (IC50 ca. 40 microM), the Ab-catalyzed reaction was completely inhibited at less than 1 microM of the TSA. The relative rates and efficiencies of the MAb-catalyzed 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.
authors.affiliation: Istituto di Chimica degli Ormoni, CNR, Milano, Italy.
authors.forename: S
authors.initials: S
authors.lastname: Riva
authors.forename: M
authors.initials: M
authors.lastname: Mendozza
authors.forename: G
authors.initials: G
authors.lastname: Carrea
authors.forename: P
authors.initials: P
authors.lastname: Chattopadhyay
authors.forename: A
authors.initials: A
authors.lastname: Tramontano
mesh_terms: D000818:Animals; D017151:Antibodies, Catalytic / Q000378:metabolism*; D002384:Catalysis; D015195:Drug Design; D005260:Female; D006241:Haptens / Q000276:immunology / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008095:Lithocholic Acid / Q000031:analogs & derivatives* / Q000276:immunology / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D008958:Models, Molecular; D010755:Organophosphates / Q000276:immunology* / Q000378:metabolism*; D012710:Serum Albumin, Bovine / Q000378:metabolism*; D013329:Structure-Activity Relationship
chemical_list: C118854:3-O-(4-nitrophenylphosphate)lithocholic acid; D017151:Antibodies, Catalytic; D006241:Haptens; D010755:Organophosphates; D012710:Serum Albumin, Bovine; D008095:Lithocholic Acid