18
because some of them are specific (e.g., restriction enzymes),
peptidases are never truly specific, and the substrate used often
changes from experiment to experiment. For instance, we use pep-
tidases to screen peptide/inhibitor libraries, test various recombi-
nant substrates, or search for new substrates by proteomics. In fact,
knowing the molar amount of active sites permits the comparison
of the proteolysis efficacy of several enzymes for a given substrate.
Furthermore, the titer of an enzyme preparation does not vary
with the inhibitor used, nor does it change based on the substrate
employed in the titration procedure. Thus, enzyme titration is a
cornerstone of the biochemical analysis of enzyme kinetics.
Active site titration of a caspase is a straightforward procedure
once one understands the underlying principles. A titration reac-
tion is a two-step procedure. First, an unknown concentration of
caspase (estimated by protein quantification) is incubated with a
known concentration of titrant. This titrant must be a covalent
inhibitor that reacts stoichiometrically with its target. For caspases,
the fluoromethyl ketone irreversible inhibitor Z-VAD-fmk is per-
fectly suited. Z-VAD-fmk forms a thioether adduct with the cata-
lytic cysteine, thus irreversibly inhibiting the peptidase. Given
enough time, every caspase active site will react with an inhibitor
molecule in a 1:1 ratio until no more inhibitor is available.
Therefore, if a series of Z-VAD-fmk samples, covering a range of
concentrations that are below and above the effective caspase con-
centration, is incubated with an unknown amount of caspase, the
lowest concentration of inhibitor that inactivates 100 % of the pep-
tidase would be close to the active site concentration. The second
step of the assay is evaluation of the fraction of uninhibited enzyme
using a simple enzymatic assay. Plotting the reaction rates obtained
against the Z-VAD-fmk concentration results in an initial down-
ward straight line ([I] < [E 0 ]) that intercepts the x-axis at a value
that is equal to the concentration of peptidase ([I] = [E 0 ]). The
second portion of the graph is a flat line representing values of
inhibitor concentration that are higher than that of the enzyme
([I] > [E 0 ]). Figure 4 shows the experimental data for the active site
titration of wild-type caspase-7. Refer to the figure legend for an
explanation. An example of the calculation done to obtain the
enzyme titer is presented in Fig. 4d.
Because titration occurs in the first reaction (inhibitor plus cas-
pase), the only enzymatic requirement for the second step of the
assay (residual uninhibited caspase plus substrate) is that the hydro-
lysis rate of the reporter substrate remains constant over the
measurement period. Additionally, because the goal of the second
step is to report the fraction of uninhibited peptidase, it is not nec-
essary to know the kinetic parameters of the enzyme for the sub-
strate used. Furthermore, the concentration of substrate does not
matter as long as no significant substrate depletion occurred.Dave Boucher et al.http://www.ebook3000.com