Distinct mechanisms of antibody-mediated enzymatic reactivation in beta-galactosidase molecular sensors.
The antibody-mediated reactivation of engineered Escherichia coli beta-galactosidases [Benito et al. (1996) J. Biol. Chem. 271, 21251-21256] has been thoughtfully investigated in three recombinant molecular sensors. Proteins M278VP1, JX772A and JX795A display the highly antigenic G-H loop peptide segment of foot-and-mouth disease virus VP1 protein, accommodated in different solvent-exposed loops of the assembled tetramer. These chimaeric enzymes exhibit a significant increase in enzymatic activity upon binding of either monoclonal antibodies or sera directed against the inserted viral peptide. In JX772A but not in M278VP1, the Fab 3E5 antibody fragment promotes reactivation to the same extent as the complete antibody. On the other hand, M278VP1 Km is reduced by more than 50% in the presence of activating serum, this parameter remains invariable in JX772A and it is only slightly modified in JX795A. In these last two proteins, significant k(cat) variations can account for the increased enzymatic activity. Alternative reactivation mechanisms in the different beta-galactosidase probes are discussed in the context of the bacterial enzyme structure and its tolerance to antibody-induced conformational modifications.
Feliu
J X
JX
Institut de Biologia Fonamental and Department de Genètica i Microbiologia, Universitat Autònoma de Barcelona, Bellaterra, Spain.
Ramírez
E
E
Villaverde
A
A
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D001666:Binding Sites, Antibody; D015374:Biosensing Techniques; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology; D006168:Guinea Pigs; D007106:Immune Sera / Q000494:pharmacology; D007140:Immunoglobulin Fab Fragments / Q000494:pharmacology; D007700:Kinetics; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D001616:beta-Galactosidase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D007106:Immune Sera; D007140:Immunoglobulin Fab Fragments; D011993:Recombinant Fusion Proteins; D001616:beta-Galactosidase