Caspases,Paracaspases, and Metacaspases Methods and Protocols

14

Kinetic parameter determination is a very useful method to com-

pare caspase activity based on their primary specificities. Therefore,

it is highly recommended that every caspase preparation is charac-

terized using a suitable substrate (Table 2 ). This criterion will

ensure the quality of the caspase preparation and allow for the

identification of problems that may have occurred during the puri-

fication process that may interfere with future experiments.

Furthermore, it is essential to compare the enzymatic properties of

caspase mutants to the wild-type enzyme. Too often, mutant cas-

pases are used in cellular experiments, and conclusions are drawn

without taking into account the possibility that kinetic properties

of the mutated enzyme have changed (see Note 18).

Two fundamental parameters characterize an enzyme: the KM

and the kcat. To be brief and by assuming that the enzyme follows

or is well represented by the mechanism of Eq. 1 , KM (in molarity)

compounds the dissociation rates, k−1 and k 2 , of the enzyme–

substrate complex and its association rate k 1 (Eq. 1 ): KM equals

(k−1 + k 2 )/k 1.

ES

k

k

ES

k

+®EP+

1

1

2



(1)

Therefore, KM represents more than the affinity of an enzyme

for a substrate because it also takes into account the conversion of

that substrate into products and not only the association and dis-

sociation of the enzyme-substrate complex into free enzyme and

free substrate. However, describing KM as an affinity constant

makes it more palatable. The parameter kcat is the catalytic constant,

also referred to the turnover rate. For enzymes such as caspases, for

which a mechanism is well represented by Eq. 1 , kcat is similar to k 2.

The kcat represents the number of molecules per second converted

from the enzyme–substrate complex into the free enzyme and the

products.

Without going into the mathematics and theory, the rate of

product formation dP/dt or v for Eq. 1 can be represented by the

Michaelis–Menten equation that is often written as:

d

d M

P

t

v

VS

KS

==

[]

+[]

max

(2)

Vmax is the rate of product formation when the enzyme is satu-

rated, and equals kcat[E]. Thus, Vmax is the maximal rate of product

generation by a specific amount of enzyme. Vmax is used when the

concentration of active enzyme is unknown, such as in an extract

during purification procedures. One of the most revealing aspect

of this equation is that when KM = [S], v = 1/2Vmax. This is why KM

reflects the concentration of substrate at which the enzymatic reaction

3.2 Enzymatic
Characterization
of Caspases

Dave Boucher et al.

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