Mechanism of inactivation of a catalytic antibody by p-nitrophenyl esters.
Antibody CNJ206 catalyses the hydrolysis of p-nitrophenyl esters with significant rate enhancement; however, after a few cycles, 90% of the catalytic activity of CNJ206 is irreversibly lost. This report investigates the properties of the inactivated Fab (fragment antigen binding). After inactivation, the residual esterase activity of CNJ206 is similar to that of the catalytic antibody inhibited by the transition-state analogue (TSA) used to elicit it; the affinity of CNJ206 for the TSA is also dramatically lowered. Here we propose a simple scheme that accounts for the steady-state kinetics of inactivation. The following lines of evidence, when taken together, suggest that stable acylated tyrosine side chains within or close to the Fab combining site are involved in the inactivation process: isoelectric focusing and matrix-assisted-laser-desorption-ionisation-time-of-flight (MALDI-TOF) mass spectrometry show that incubation with substrate results in several acylated Fab species; inactivation is stable at pH 8, is reversed by mild hydroxylamine treatment and follows the same kinetics as inhibition of binding, which is slowed down by the presence of the TSA hapten. Analysis of the Fab-TSA X-ray structure shows that three tyrosine residues are potential candidates for the inactivation of CNJ206 by its substrates, Tyr L96 being the most likely one; this also suggests that site-directed mutation of one or more of these residues might prevent substrate inactivation and significantly improve catalysis.
Gigant
B
B
Laboratoire d'Enzymologie et Biochimie Structurales, UPR 9063 CNRS, Gif-sur-Yvette, France.
Charbonnier
J B
JB
Golinelli-Pimpaneau
B
B
Zemel
R R
RR
Eshhar
Z
Z
Green
B S
BS
Knossow
M
M
D017151:Antibodies, Catalytic / Q000737:chemistry / Q000187:drug effects*; D002384:Catalysis; D018360:Crystallography, X-Ray; D004952:Esters / Q000494:pharmacology; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry / Q000187:drug effects*; D007525:Isoelectric Focusing; D007700:Kinetics; D008969:Molecular Sequence Data; D009596:Nitrophenols / Q000737:chemistry / Q000494:pharmacology*; D011487:Protein Conformation; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D017151:Antibodies, Catalytic; D004952:Esters; D007140:Immunoglobulin Fab Fragments; D009596:Nitrophenols