The three-dimensional structures of cystatins, and
other evidence, suggest that the flexible N-terminal
region of these inhibitors may bind to target proteinases
independent of the two rigid hairpin loops forming the
remainder of the inhibitory surface. In an attempt to demonstrate
such two-step binding, which could not be identified in
previous kinetics studies, we introduced a cysteine residue
before the N-terminus of cystatin A and labeled this residue
with fluorescent probes. Binding of AANS- and AEDANS-labeled
cystatin A to papain resulted in ∼4-fold and 1.2-fold
increases of probe fluorescence, respectively, reflecting
the interaction of the N-terminal region with the enzyme.
Observed pseudo-first-order rate constants, measured by
the loss of papain activity in the presence of a fluorogenic
substrate, for the reaction of the enzyme with excess AANS-cystatin
A increased linearly with the concentration of the latter.
In contrast, pseudo-first-order rate constants, obtained
from measurements of the change of probe fluorescence with
either excess enzyme or labeled inhibitor, showed an identical
hyperbolic dependence on the concentration of the reactant
in excess. This dependence demonstrates that the binding
occurs in two steps, and implies that the labeled N-terminal
region of cystatin A interacts with the proteinase in the
second step, subsequent to the hairpin loops. The comparable
affinities and dissociation rate constants for the binding
of labeled and unlabeled cystatin A to papain indicate
that the label did not appreciably perturb the interaction,
and that unlabeled cystatin therefore also binds in a similar
two-step manner. Such independent binding of the N-terminal
regions of cystatins to target proteinases after the hairpin
loops may be characteristic of most cystatin–proteinase
reactions.