Substrate-dependent modulation of enzyme activity by allosteric effector antibodies.
We investigate the kinetic effects of antibody variable domain fragments derived from heavy chain antibodies (VHH domains) that behave as allosteric effectors of the nucleoside hydrolase from Trypanosoma vivax (TvNH). Strikingly, these antibodies can stimulate or inhibit TvNH steady-state activity, depending on the substrate used. This effect was investigated in greater detail using steady-state and pre-steady-state kinetic experiments. The most potent allosteric effector, VHH 1589, inhibits certain steps on the TvNH catalytic pathway (e.g. N-glycosidic bond cleavage) but increases the rates of others (e.g. substrate and product release). For the natural nucleoside 7-methyl guanosine, where product ribose release is rate determining, the net effect of VHH 1589 binding is to increase k(cat). For the poor substrate pNPR, VHH 1589 causes chemistry (O-glycosidic bond cleavage) to become rate determining and both k(cat)/K(m) and k(cat) to decrease. Thus, the substrate-dependent effects of VHH 1589 binding are caused by differences in the relative rates of chemistry with respect to subsequent steps on the catalytic pathway for these two substrates. We discuss possible mechanisms for these kinetic effects and the implications for allosteric effector drug development.
Structural Biology, Free University of Brussels, Brussels, Belgium. jbarlow@vub.ac.be
John N
JN
Barlow
Katja
K
Conrath
Jan
J
Steyaert
D000494:Allosteric Regulation; D000818:Animals; D002384:Catalysis; D007135:Immunoglobulin Variable Region / Q000494:pharmacology*; D007700:Kinetics; D009699:N-Glycosyl Hydrolases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010446:Peptide Fragments / Q000494:pharmacology; D017434:Protein Structure, Tertiary; D011684:Purine Nucleosides / Q000494:pharmacology; D011744:Pyrimidinones / Q000494:pharmacology; D016689:Trypanosoma vivax / Q000201:enzymology
D007135:Immunoglobulin Variable Region; D010446:Peptide Fragments; D011684:Purine Nucleosides; D011744:Pyrimidinones; C113101:forodesine; D009699:N-Glycosyl Hydrolases