Detection of a catalytic antibody species acylated at the active site by electrospray mass spectrometry.
The chemical interactions between a catalytic antibody Fv fragment and ester substrates were examined using pneumatically assisted electrospray (ion spray) mass spectrometry. Upon addition of the p-nitrophenyl ester substrate to the antibody fragment, an antibody fragment species that represents approximately 8% of the total Fv concentration is clearly observed in the electrospray spectrum. The observed increase in molecular weight of the Fv fragment corresponds to the mass of the acyl group of the substrate. Formation of the acyl-Fv species is blocked by preincubation of the antibody fragment with hapten inhibitor, suggesting that the acyl linkage involves a residue in the active site of the antibody. The acyl-Fv species is not observed when the corresponding p-chlorophenyl ester substrate is used, indicating that the level of this species is dependent on the leaving group of the substrate. The acylated species is not observed for a site-directed mutant lacking catalytic activity, His L91 Gln. The present results are consistent with modeling studies of the structure of the Fv fragment and provide strong confirmatory evidence for the multistep kinetic mechanism previously proposed for this antibody.
Krebs
J F
JF
Department of Molecular Biology, Scripps Research Institute, La Jolla, California 92037.
Siuzdak
G
G
Dyson
H J
HJ
Stewart
J D
JD
Benkovic
S J
SJ
D000215:Acylation; D000813:Anilides / Q000378:metabolism; D017151:Antibodies, Catalytic / Q000737:chemistry*; D001665:Binding Sites; D007128:Immunoglobulin Fragments / Q000378:metabolism; D013058:Mass Spectrometry / Q000379:methods; D011994:Recombinant Proteins / Q000378:metabolism
D000813:Anilides; D017151:Antibodies, Catalytic; D007128:Immunoglobulin Fragments; D011994:Recombinant Proteins; C055966:immunoglobulin Fv