J. Biol. Chem., Vol. 264, Issue 14, 8004-8011, 05, 1989
Inhibition by bestatin of a mouse ascites tumor dipeptidase. Reversal by certain substrates
EK Patterson
Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111.
Bestatin, [(2S,3R)-3-amino-2-hydroxy-4-phenyl-butanoyl]-L-leucine, a known
inhibitor of aminopeptidases, is shown to be a potent linear competitive
inhibitor (KI,2.7 nM) of a dipeptidase purified from Ehrlich-Lettre
hyperdiploid mouse ascites tumor cells. This inhibition can be classified
as "slow binding" but not as "tight binding." Substrate protects the enzyme
from bestatin inhibition when enzyme and inhibitor are in approximately
equimolar concentrations. Addition of substrate (6 mM) partially (by about
20%) reverses dipeptidase inhibition by bestatin, but the time required for
maximum recovery depends on the nature of the substrate. Substrates with
lower Km (0.28- 1.4 mM) values that exhibit substantial substrate
inhibition require longer times (23-65 min) than those with higher Km
values that show little substrate inhibition. Substrates with Km values
higher than 1.5 mM do not reverse inhibition. The inhibition of the tumor
dipeptidase by bestatin has been compared with inhibition by a variety of
inhibitors of other Zn-metallo-proteolytic enzymes. These inhibitors were
far less potent (KI, 0.063-10 mM), indicating a difference between the
tumor dipeptidase and other enzymes of that class. Our results are
discussed in terms of a postulated model of the bestatin molecule in the
active site of the tumor dipeptidase, an enzyme which has not been studied
by x-ray crystallographic means. The phenyl group of bestatin is placed in
a hydrophobic pocket that is external but adjacent to the active site of
the tumor dipeptidase. The shape of this pocket, as it appears from our
results plus modeling, is such that only certain R groups of substrate can
fit. The existence of such a pocket might explain the differential effect
of substrates in the reversal of bestatin inhibition of the dipeptidase and
also might explain substrate inhibition by misalignment of R groups into
this pocket.
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