22
Many in vitro methods enable the assessment of specificity. However,
one must be careful in using the primary specificity of a caspase to
assess its preference on natural substrates. The recent report that
caspase determinants located outside the substrate- binding pocket
can overcome a poor cleavage site motif demonstrates the impor-
tance of studying protein substrates [ 32 ]. Practically, the determina-
tion of kinetic parameters for a protein, as described in the previous
subheading, is difficult. First, it is problematic to set up assays with
protein substrate concentrations above KM (e.g., >10 μM). Second,
not all protein substrates can be produced as recombinant proteins
or purified to homogeneity from tissues or cells.
In pseudo-first order conditions ([S] ≪ KM), Eq. 2 can be
simplified to v ≈ Vmax[S]/KM. The proportion of substrate used by
an enzyme is described by Eq. 3 , which can be rearranged to
Eq. 4 [ 25 ]:p
=-1e-k[]Et
(3)
kp=-ln()^1 - /[]Et (4)
where p is the proportion of substrate cleaved (varies between 0
and 1) by the enzyme [E] at time t in seconds. The parameter k
approximates kcat/KM. However, unless pseudo-first order condi-
tions are met, it is better to refer to k as kcat,app/KM,app. It is noted
that the substrate does not appear in Eqs. 3 or 4. Thus, it is not
necessary to know the protein substrate concentration to estimate
kcat/KM in pseudo-first order conditions.
Another interesting aspect of pseudo-first order kinetics is that
it is possible to use a mixture of proteins, such as a cell lysate, as a
source of substrate. Importantly, cell lysate contains other sub-
strates. However, the competition for the enzyme will be negligi-
ble as long as those other substrates, as a whole, also meet
pseudo-first order conditions. This statement is valid because the
rate of hydrolysis of a substrate in the presence of a competitor
substrate S′, which has a Michaelis–Menten’s constant KM′, is:d
d
M
MP
tvVSKS
KS==[]+æ []
èçö
ø÷+[]max1¢
¢(5)If S′ is much smaller than KM′ as in a sufficiently diluted lysate,
the denominator approaches KM + [S], and the enzyme behaves as
if no other substrate were present. Thus, this condition allows the
estimation of kinetic parameters from a cellular lysate using Eq. 3.
Figure 5 shows the analysis of poly(ADP ribose) polymerase 1
(PARP-1) cleavage in a cell lysate by caspase-3 and caspase-7.3.3 Studying Protein
Caspase Substrates
Dave Boucher et al.http://www.ebook3000.com