Mechanistically different catalytic antibodies obtained from immunization with a single transition-state analog.
The variable-region peptide sequence and steady-state kinetic behavior are compared for a family of catalytic antibodies that arose from the same immune response to a transition-state analog. The crystal structure of the most catalytically active member of the family (17E8) has been solved to 2.5 A resolution and shows that the antibody active site contains a SerH99-HisH35 (H = heavy chain) catalytic dyad analogous to the Ser-His-Asp catalytic triad of serine proteases. The variable-region peptide sequence of the next most active antibody (29G11) differs from that of 17E8 by nine heavy-chain point mutations, and results from computer modeling suggest that the three-dimensional structure of 29G11 is similar to that of 17E8. In addition, 29G11 is an efficient catalytic antibody; it possesses 26% of the hydrolytic activity of 17E8. There is one active-site mutation in 29G11 compared to 17E8; position 99 of the heavy chain of 29G11 contains a glycine residue in place of the nucleophilic serine at this position in 17E8. Consistent with this mutation, results from pH-rate studies and hydroxylamine partitioning experiments indicate that in contrast to the catalytic mechanism of 17E8, the mechanism of 29G11-catalyzed esterolysis does not feature nucleophilic catalysis.
Guo
J
J
Department of Pharmaceutical Chemistry, University of California, San Francisco 94143-0446.
Huang
W
W
Zhou
G W
GW
Fletterick
R J
RJ
Scanlan
T S
TS
D000595:Amino Acid Sequence; D000818:Animals; D017151:Antibodies, Catalytic; D000911:Antibodies, Monoclonal / Q000378:metabolism*; D001665:Binding Sites; D001666:Binding Sites, Antibody; D018360:Crystallography, X-Ray; D004950:Esterases / Q000737:chemistry*; D006241:Haptens; D006863:Hydrogen-Ion Concentration; D007140:Immunoglobulin Fab Fragments; D007135:Immunoglobulin Variable Region; D066298:In Vitro Techniques; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D009646:Norleucine / Q000737:chemistry; D011487:Protein Conformation; D013329:Structure-Activity Relationship
D017151:Antibodies, Catalytic; D000911:Antibodies, Monoclonal; D006241:Haptens; D007140:Immunoglobulin Fab Fragments; D007135:Immunoglobulin Variable Region; D009646:Norleucine; D004950:Esterases