15is at half its maximum rate. One can also realize that it becomes
difficult to saturate an enzyme if KM is relatively high, i.e., 10 KM
results in 0.91 Vmax.
Finally, a useful term is kcat/KM (units of M−1 s−1), which is the
catalytic specificity of the enzyme. This term takes into account
both KM and kcat. The catalytic specificity is a parameter that can be
obtained even if both kcat and KM cannot be derived individually
(see Subheading 3.3). The catalytic specificity is the best way to
compare enzyme efficacy for various substrates and to compare
enzymes in general.
To determine kcat, one must determine the active enzyme con-
centration, which can be achieved by titration. Unfortunately, too
many studies neglect this important step in caspase substrate char-
acterization. Basing caspase concentration on protein quantity
(e.g., μg/mL) or enzyme units (e.g., units/mL) should be dis-
couraged because neither measurement reflects the active enzyme,
but rather an amount of protein, active or not, or a quantity of
enzymatic work on a standard substrate, respectively.
Many studies use peptidic substrates to characterize caspase
activity. One of the most prevalent misconceptions is that many
assume that the preferred recognition motif is specific for a given
caspase (e.g., DEVD for caspase-3) [ 4 , 5 , 7 ]. This assumption is false
if one understands how most peptidases, including caspases, work
and how the preferred motifs were determined in the first place.
Furthermore, this notion has been disproven experimentally [ 23 ].
Because caspases recognize their substrates’ cleavage sites primarily
via four subsites (S1 to S4; Schechter–Berger nomenclature [ 24 ]),
tetrapeptides are used. Interestingly, caspase-2 has a substrate-bind-
ing pocket that extends to recognize at least five residues, and thus,
pentapeptides are used for this enzyme [ 25 ]. To track hydrolysis, the
peptide is linked at its C-terminus to a leaving group. Usually, this
group is fluorescent, such as Afc (7-amino- 4-trifluoromethyl-
coumarin) or Amc (7-amino-4- methylcoumarin), or is a colored
compound such as p-nitroaniline (pNa). Cleavage of the peptide
releases the fluorophore or the chromophore and enables the mea-
surement of caspase activity with a spectrofluorometer (Afc and
Amc) or a spectrophotometer (pNa) plate reader, respectively. It is
important to note that several other substrates are available, but
many of them have two peptides attached to the fluorophore [e.g.,
(Z-DEVD) 2 -Rh110 or MR-(DEVD) 2 ]. Although many of these
substrates are sensitive, their hydrolysis reactions do not conform to
the mechanism depicted in Eq. 1 , so the product resulting from the
cleavage is still a substrate for the enzyme. Therefore, these dual-
peptide substrates are not suitable for enzymatic characterization
using the traditional Michaelis–Menten equation.
This section describes procedures to quantify the active site
concentration in a caspase preparation and to determine the funda-
mental kinetic parameters of caspases. When all reagents are available
Apoptotic Caspases Assays