52
C.B. Elias and J.B. Joshi
and series deactivation mechanisms. Henley and Sadana [17] proposed a unified general model which provides an insight into the enzyme structure and
function and helps in determining the deactivation pattern which follows.
The model considers that deactivation follows a multistep process, occurring
as a series, in parallel or both, by which the native enzyme, E, is altered to some
intermediate forms, Ex, E2 ..., E,. Each of these may be deactivated to a final
dead form Ed. Considering the general scheme of deactivation including a series
and parallel deactivation, the total enzymatic activity was expressed as
a = Cle -i't + C2 e-z2t + ... + C,e -z"t
(6)
where Ci are the constants of integration and 2i is a function of the forward and
reverse first order rate and the first order deactivation rate constants. This
general equation may be used to determine the number of steps required to
represent the mechanism of a given deactivation process [17]. Each exponential
term here represents an independent, potentially active form of the enzyme. If
there is a rapid equilibrium between any two forms then only one of them is
independent, as the concentration of all forms involved is governed by the
equilibrium relationship. This has been used to explain deactivation of rabbit
skeletal muscle AMP deaminase due to deprotonation of the active site [18].
Some exceptions to this model are deactivation due to higher order processes
such as autolysis especially of proteolytic enzymes [19] and biological contamination.
Complex non-first order kinetics are displayed by various enzymes. These
have been classified into two types.
1) a biphasic behaviour where a rapid inactivation is followed by a decelerated
decay finally resulting in an activity plateau
2) a "grace period behaviour" where an initial stable phase of little activity loss
is followed by periods of accelerating and decelerating inactivation [20].
Enzymes such as luciferase and acid phosphatase show biphasic behaviour
[21-22] whereas galactosyl transferase and fl-D-fructofuranosidase show the
latter type of behaviour [23, 24]. Lencki et al. [20] described the effects of
subunit dissociation, denaturation, aggregation, coagulation and decomposition on enzyme inactivation kinetics, and distinguished the reversible and
irreversible mechanisms of deactivation.
The native to denatured structural transition of globular monomeric proteins is considered to be a reversible process. The presence of a stable intermediate during the transition process could lead to the observed grace period
behaviour or to two first-order slopes i.e. biphasic behaviour, and has been
suggested as the source of non-first order kinetics of enzyme inactivation
kinetics [25]. However, these intermediates are generally observed only at very
low concentrations relative to the native and denatured protein species and the
time frame of these transitions is very short relative to the protein deactivation
time and hence these may be considered to be in equilibrium and thus do not
directly affect the enzyme kinetics. Many enzymes are olimeric comprising
C.B. Elias and J.B. Joshi
and series deactivation mechanisms. Henley and Sadana [17] proposed a unified general model which provides an insight into the enzyme structure and
function and helps in determining the deactivation pattern which follows.
The model considers that deactivation follows a multistep process, occurring
as a series, in parallel or both, by which the native enzyme, E, is altered to some
intermediate forms, Ex, E2 ..., E,. Each of these may be deactivated to a final
dead form Ed. Considering the general scheme of deactivation including a series
and parallel deactivation, the total enzymatic activity was expressed as
a = Cle -i't + C2 e-z2t + ... + C,e -z"t
(6)
where Ci are the constants of integration and 2i is a function of the forward and
reverse first order rate and the first order deactivation rate constants. This
general equation may be used to determine the number of steps required to
represent the mechanism of a given deactivation process [17]. Each exponential
term here represents an independent, potentially active form of the enzyme. If
there is a rapid equilibrium between any two forms then only one of them is
independent, as the concentration of all forms involved is governed by the
equilibrium relationship. This has been used to explain deactivation of rabbit
skeletal muscle AMP deaminase due to deprotonation of the active site [18].
Some exceptions to this model are deactivation due to higher order processes
such as autolysis especially of proteolytic enzymes [19] and biological contamination.
Complex non-first order kinetics are displayed by various enzymes. These
have been classified into two types.
1) a biphasic behaviour where a rapid inactivation is followed by a decelerated
decay finally resulting in an activity plateau
2) a "grace period behaviour" where an initial stable phase of little activity loss
is followed by periods of accelerating and decelerating inactivation [20].
Enzymes such as luciferase and acid phosphatase show biphasic behaviour
[21-22] whereas galactosyl transferase and fl-D-fructofuranosidase show the
latter type of behaviour [23, 24]. Lencki et al. [20] described the effects of
subunit dissociation, denaturation, aggregation, coagulation and decomposition on enzyme inactivation kinetics, and distinguished the reversible and
irreversible mechanisms of deactivation.
The native to denatured structural transition of globular monomeric proteins is considered to be a reversible process. The presence of a stable intermediate during the transition process could lead to the observed grace period
behaviour or to two first-order slopes i.e. biphasic behaviour, and has been
suggested as the source of non-first order kinetics of enzyme inactivation
kinetics [25]. However, these intermediates are generally observed only at very
low concentrations relative to the native and denatured protein species and the
time frame of these transitions is very short relative to the protein deactivation
time and hence these may be considered to be in equilibrium and thus do not
directly affect the enzyme kinetics. Many enzymes are olimeric comprising