Mechanistic studies of a tyrosine-dependent catalytic antibody.
A pre-steady-state multiple-turnover kinetic burst is observed during hydrolysis of phenyl acetate by the catalytic antibody, 20G9. The burst is caused by partial product inhibition by phenol (Ki,app = 2.5 microM), which lowers both kcat and KM by almost an order of magnitude without affecting kcat/KM. The acid limb of the steady-state kcat pH profile of native 20G9 has a pKa of 9.6, suggesting a catalytic role for tyrosine. Additional evidence for an essential tyrosyl residue is that mild treatment of 20G9 with tetranitromethane nitrates a single tyrosine per equivalent of antigen binding sites and the mononitrated derivative has less than 5% of the native activity. Near-UV absorbance spectroscopy suggests that the alternative substrates N-carbobenzoxyglycine O-phenyl ester (ZG-OPh) and N-acetylglycine O-phenyl ester (AcG-OPh) acylate multiple tyrosines on the antibody. Neither ZG-OPh nor AcG-OPh are measurably catalyzed once appreciable acylation has taken place. Antibody acylated by ZG-OPh is inactive toward phenyl acetate hydrolysis, but can be reactivated by hydroxylamine. The data and derived kinetic rate equations are consistent with an acyl mechanism for phenyl acetate hydrolysis in which phenol inhibits by binding to a covalent O-acetyltyrosyl intermediate, slowing deacylation. Although the data are consistent with such a mechanism, they do not rule out other plausible, yet less unifying mechanisms of phenol inhibition; the observed burst could conceivably result from partial mixed phenol inhibition or from phenol-induced nonproductive substrate binding. Because antibodies often use tyrosines in antigen binding, tyrosyl catalytic antibodies may be commonly encountered in the future.
Martin
M T
MT
IGEN, Inc., Rockville, Maryland 20852.
Napper
A D
AD
Schultz
P G
PG
Rees
A R
AR
D000911:Antibodies, Monoclonal / Q000737:chemistry*; D001202:Ascitic Fluid / Q000737:chemistry; D001666:Binding Sites, Antibody; D001667:Binding, Competitive; D002384:Catalysis; D006868:Hydrolysis; D007700:Kinetics; D013329:Structure-Activity Relationship; D014443:Tyrosine / Q000276:immunology*
D000911:Antibodies, Monoclonal; D014443:Tyrosine