Food Chemistry

118 2 Enzymes

termediary enzyme-substrate complex, EA. The
complex then forms the product P and releases
the free enzyme:

(2.30)

In order to determine the catalytic activity of the
enzyme, the decrease in substrate concentration
or the increase in product concentration as a func-
tion of time can be measured. The activity curve
obtained (Fig. 2.21) has the following regions:

a) The maximum activity which occurs for

a few msec until an equilibrium is reached

between the rate of enzyme-substrate

formation and rate of breakdown of this

complex.

Measurements in this pre-steady state region

which provide an insight into the reaction

steps and mechanism of catalysis are difficult

and time consuming. Hence, further analysis

fo the pre-steady state will be ignored.

b) The usual procedure is to measure the en-

zyme activity when a steady state has been

reached. In the steady state the intermedi-

ary complex concentration remains constant

while the concentration of the substrate and

end product are changing. For this state, the

following is valid:

dEA

dt

=−

dEA

dt

(2.31)

c) The reaction rate continuously decreases in

this region in spite of an excess of substrate.

The decrease in the reaction rate can be con-

sideredtobearesultof:

Enzyme denaturation which can readily oc-

cur, continuously decreasing the enzyme con-

centration in the reaction system, or the prod-

uct formed increasingly inhibits enzyme ac-

tivity or, after the concentration of the prod-

uct increases, the reverse reaction takes place,

converting the product back into the initial re-

actant.

Since such unpredictable effects should be
avoided during analysis of enzyme activities, as
a rule the initial reaction rate, v 0 , is measured as
soon as possible after the start of the reaction.
The basics of the kinetic properties of enzymes
in the steady state were given byBriggsand

Fig. 2.21.Progress of an enzyme-catalyzed reaction

Haldane (1925) and are supported by earlier

mathematical models proposed byMichaelisand

Menten(1913).

The following definitions and assumptions should

be introduced in relation to the reaction in Equa-

tion 2.30:

[E 0 ]=total enzyme concentration available at

the start of the catalysis.

[E] =concentration of free enzyme not bound

to the enzyme-substrate complex, EA,

i. e. [E]=[E 0 ]−[EA].

[A 0 ]=total substrate concentration available at

the start of the reaction. Under these con-

ditions, [A 0 ] [E 0 ]. Since in catalysis

only a small portion of A 0 reacts, the

substrate concentration at any time, [A],

is approximately equal to [A 0 ].

When the initial reaction rate, v 0 , is considered,

the concentration of the product, [P], is 0. Thus,

the reaction in Equation 2.30 takes the form:

dP

dt

=υ 0 =k 2 (EA) (2.32)

The concentration of enzyme-substrate complex,

[EA], is unknown and can not be determined ex-

perimentally for Equation 2.32. Hence, it is cal-

culated as follows: The rate of formation of EA,

according to Equation 2.30, is:

dEA

dt

=k 1 (E)(A 0 ) (2.33)

and the rate of EA breakdown is:

−

dEA

dt

=k− 1 (EA)∗k 2 (EA) (2.34)