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 investi gates the properties of the inactivated Fab (fragment antigen binding) . After inactivation, the residual esterase activity of CNJ206 is simi lar to that of the catalytic antibody inhibited by the transition-stat e analogue (TSA) used to elicit it; the affinity of CNJ206 for the TSA is also dramatically lowered; Here we propose a simple scheme that ac counts for the steady-state kinetics of inactivation. The following li nes of evidence, when taken together, suggest that stable acylated tyr osine side chains within or close to the Fab combining site are involv ed in the inactivation process: isoelectric focusing and ssisted-laser -desorption-ionisation-time-of-flight (MALDI-TOF) mass spectrometry sh ow that incubation with substrate results in several acylated Fab spec ies; inactivation is stable at pH 8, is reversed by mild hydroxylamine treatment and follows the same kinetics as inhibition of binding, whi ch is slowed down by the presence of the TSA hapten. Analysis of the F ab-TSA X-ray structure shows that three tyrosine residues are potentia l candidates for the inactivation of CNJ206 by its substrates, Tyr L96 being the most likely one; this also suggests that site-directed muta tion of one or more of these residues might prevent substrate inactiva tion and significantly improve catalysis.