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To probe the mechanism of the catalytic antibody NPN43C9, we have constructed a
three-dimensional model of the NPN43C9 variable region using our antibody
structural database (ASD), which takes maximal advantage of immunoglobulin
sequence and structural information. The ASD contains separately superimposed
variable light and variable heavy chains, which reveal not only conserved
backbone structure, but also structurally conserved side-chain conformations.
The NPN43C9 model revealed that the guanidinium group of light chain Arg L96 was
positioned at the bottom of the antigen-binding site and formed a salt bridge
with the antigen's phosphonamidate group, which mimics the negatively charged,
tetrahedral transition states in the hydrolysis reaction. Thus, the model
predicts both binding and catalytic functions for Arg L96, which previously had
not been implicated in either. First, Arg L96 should enhance antigen binding by
electrostatically complementing the negative charge of the antigen, which is
buried upon complex formation. Second, Arg L96 should promote catalysis by
electrostatically stabilizing the negatively charged transition states formed
during catalysis. These hypotheses were tested experimentally by design and
characterization of the R-L96-Q mutant, in which Arg L96 was replaced with Gln
by site-directed mutagenesis. As predicted, antigen binding in the R-L96-Q
mutant was decreased relative to that in the parent NPN43C9 antibody, but
binding of antigen fragments lacking the phosphonamidate group was retained. In
addition, the R-L96-Q mutant had no detectable esterase activity. Thus, the
computational model and experimental results together suggest a mechanism by
which the catalytic antibody NPN43C9 stabilizes high-energy transition states
during catalysis.
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