Identification of rate-controlling enzymes
The individual enzymes in a metabolic pathway combine to produce a given flow of
substrates and of products through the pathway. This flow is referred to as themetabolic
flux. Its value is determined by factors such as the availability of starting substrate and
cofactors but above all by the activity of the individual enzymes. Studies have revealed
that the enzymes in a given pathway do not all possess the same activity. As a conse-
quence, one or at most a small number with the lowest activity determine the overall flux
through the pathway. In order to identify theserate-controlling enzymesthree types of
study need to be carried out:- in vitrokinetic studies of each individual enzyme conducted under experimental
conditions as near as possible to those foundin vivoand such that the enzyme is
saturated with substrate (i.e. such that [S]> 10 Km); - studies to determine whether or not each individual enzyme stage operates at or near
equilibriumin vivo; - studies to determine theflux control coefficient,C,for each enzyme. This is a property
of the enzyme that expresses how the flux of reactants through a pathway is influenced
by a change in the activity (note:notconcentration) of the enzyme under the prevailing
physiological conditions. Such a change may be induced by allosteric activators or
inhibitorsor by feedback inhibition. Values forCcan varybetween 0 and1. A flux control
coefficient of 1 means that the flux through the pathway varies in proportion to the
increase in the activity of the enzyme whereas a flux control coefficient of 0 means that
the flux is not influenced by changes in the activity of that enzyme. The sum of theC
values for all the enzymes in a given pathway is 1 so that the higher a givenCvalue the
greater the impact of that enzyme on the flux through the pathway. TheseCvalues are
therefore highest for the rate-determining enzymes.
A reaction that is not at or near equilibrium and which is therefore associated with a
large free energy change, is potentially a rate-limiting enzyme since the most probable
reason for the non-establishment of equilibrium is the lack of adequate enzyme activity.
To test for a non-equilibrium reaction it is necessary to analyse the concentration of each
substrate and productin vivo. This is normally done by stopping all further reactions by
denaturing the enzymes by the addition of a suitable denaturant to thein vivotest system
and then analysing the analytes by a technique such as chromatography or NMR.
The application of these three teststo the enzymesin the glycolytic pathway shows that
three of the ten enzymes, hexokinase, 6-phosphofructokinase and pyruvate kinase, have
a potential rate-limiting role and do not achieve equilibrium but are associated with a
large negative free energy change and are therefore effectively irreversible. The same
three enzymes have the largestCvalues. Studies have revealed that all three enzymes are
subject to various control mechanisms of their activity and all contribute to the control of
flux through the glycolytic pathway. The actual quantitative values for the three test
parameters vary between cell types in a given organism and between cells of a given type
in different organisms.
Hexokinase exists in four isoenzyme forms, the first three of which are subject to
inhibition by glucose-6-phosphate, the product of the reaction. Isoenzyme IV (also617 15.5 Control of enzyme activity