Kinetic and Mechanistic Characterization of an Efficient Hydrolytic Antibody: Evidence for the Formation of an Acyl Intermediate

In this paper we report the generation and mechanistic characterization of an unusually active catalytic antibody raised to a phosphonate transition-state analog of norleucine. The antibody (17E8) catalyzes the hydrolysis of both norleucine and methionine phenyl esters and is specific for enantiomers that possess the natural 5 configuration (l) at the α-carbon. The antibody shows side-chain selectivity (k_cat/K_M) for norleucine over methionine phenyl esters and also shows selectivity for amino acid ester substrates bearing a formyl substituent at the α-amino position. The antibody-catalyzed hydrolysis of all the ester substrates exhibits a bell-shaped pH-rate profile that is consistent with a mechanistic scheme featuring two ionizable active site residues that mediate catalysis. The calculated pK_avalues for these two residues are 8.9 and 10.1, and in the most catalytically active state of the antibody, the pK_a, 8.9 residue is deprotonated and the pK_a10.1 residue is protonated. In the presence of hydroxylamine, partitioning between hydrolysis and hydroxaminolysis is observed, indicating that a covalent acyl intermediate is formed in the antibody-catalyzed reaction. The hydroxaminolysis:hydrolysis product ratio increases with increasing hydroxylamine concentration, while the overall reaction velocity (as measured by phenol release) is unaffected by increasing hydroxylamine concentration. These data support a mechanism involving a covalent acyl intermediate where formation of the intermediate is the rate-determining step in the antibody-catalyzed hydrolysis reaction.