title: Conformational changes affect binding and catalysis by ester-hydrolysing antibodies.
abstract: D2.3, D2.4 and D2.5 are ester-hydrolysing antibodies raised against a phosphonate transition state analogue (TSA). All three antibody-TSA binding kinetics, as monitored by fluorescence quenching, indicate an "induced-fit" mechanism: fast bimolecular association followed by a unimolecular isomerisation (k=1-7 s-1). Isomerisation leads to a 30-170-fold increase in affinity towards the TSA and, consequently, to higher catalytic rates. Antibody D2.3 exhibits a complex three-step binding mechanism, in which the last step is a "very slow" isomerisation (k<0.02 s-1). This very slow isomerisation is limiting the rate of catalysis by D2.3, as indicated by the kinetics of product release which show characteristics of enzyme "conformational memory" or "hysteresis". The results support a mechanism consisting of pre-equilibrium between "nether-active" (low affinity) and "active" (high affinity) antibody conformers (prior to ligand addition) as well as induced-fit, i.e. isomerisation of the nether-active ligand-antibody complex to give the active complex. Crystal structures of these antibodies, free and complexed, have previously indicated that their conformation does not change upon binding. Here, we show that the buffer used to crystallise the antibodies, and in particular its polyethylene glycol component, alters the pre-equilibrium in favour of the active conformer, leading to its crystallisation both in the presence and in the absence of the TSA.
authors.affiliation: Department of Immunology, The Weizmann Institute of Science, Rehovot, 76100, Israel.
authors.forename: A B
authors.initials: AB
authors.lastname: Lindner
authors.forename: Z
authors.initials: Z
authors.lastname: Eshhar
authors.forename: D S
authors.initials: DS
authors.lastname: Tawfik
mesh_terms: D000818:Animals; D000906:Antibodies / Q000737:chemistry* / Q000276:immunology*; D002384:Catalysis; D004952:Esters; D006868:Hydrolysis; D051379:Mice; D063065:Organophosphonates / Q000276:immunology*; D011487:Protein Conformation; D013329:Structure-Activity Relationship
chemical_list: D000906:Antibodies; D004952:Esters; D063065:Organophosphonates