Amidase antibody 312d6, obtained
against the sulfonamide hapten
4a that mimics the transition state for
hydrolysis of a distorted amide, accelerates
the hydrolysis of the corresponding
amides 1a ± 3a by a factor of 103 at pH 8.
The mechanisms of both the uncatalyzed
and antibody-catalyzed reactions were
studied. Between pH 8 and 12 the uncatalyzed
hydrolysis of N-toluoylindoles
1a and 3a shows a simple first-order
dependence on [OH], while hydrolysis
of 3a is zeroth-order in [OH] below
pH 8. The pH profile for hydrolysis of
the corresponding tryptophan amide 2a
is more complex due to the dissociation
of the zwitterion into an anion with
pKa 9.74; hydrolysis of the zwitterionic
and the anionic form of 2a both show
simple first-order dependence on
[OH]. Absence of 18O exchange between
H2
18O/18OH and the substrate, a
normal SKIE for both 1a (kH/kD1.12)
and 3a (kH/kD1.24) and the value of
the Hammett constant for hydrolysis
of p-substituted amides 3a ± e are consistent
with an ester-like mechanism in
which formation of the tetrahedral intermediate
is rate-determining and the
amine departs as anion. The 312d6-
catalyzed hydrolysis of 3a was studied
between pH 7.5 and 9, and its independence
of pH in this range indicates that
water is the reacting nucleophile. Hydrolysis
of 3a is only partially inhibited
by the sulfonamide hapten, and this
indicates that non-specific catalysis by
the protein accompanies the specific
process. Only the nonspecific process is
observed in the hydrolysis of amides 3
with para substituents other than methyl.
Binding studies on the corresponding
series of p-substituted sulfonamides
5a ± e confirm the high specificity of
antibody 312d6 for p-methyl substituted
substrates.
