Regulation of Enzyme Activity

Bryant Miles
All living beings must be able to regulate the catalytic activities of enzymes to coordinate metabolic
processes, respond to environmental changes and differentiate.
Availability
The amount of an enzyme depends upon its rate of synthesis and rate of degradation. Cells tightly
regulate both processes. We will study the regulation of gene expression later on in this semester (Time
permitting). Protein degradation is covered in 411.
There are 4 mechanisms for regulating enzyme activity.
1. Proteolysis
2. Reversible Covalent Modification
3. Allosteric Regulation
4. Isozymes
I. Proteolysis.
We have already discussed zymogens, proteins that are synthesized as
inactive molecules and activated by a specific proteolysis. This is an
irreversible activation.
Shown here is chymotrypsinogen which is activated by a single tryptic
cleavage. In chymotrypsinogen the carboxylate of Asp-194 forms a
salt bridge with His-40. After cleavage by trypsin Asp-194 forms a
new electrostatic interaction with the new N-terminal of Ile-16. This
new salt bridge allows the amide nitrogen of Gly-193 to move closer to
the amide hydrogen of Ser-195 which forms the oxy-anion hole.

II Reversible Covalent Modifications.

Regulating enzymatic activity by covalent modification of enzymes is
more widely used than proteolysis.
A common form of covalent modification is phosphorylation of side
chains of serine, threonine or tyrosine residues. The major advantage
of regulation by covalent modification is that it is reversible. The
phosphate group is introduced by a specific kinase and removed by a
specific phosphatase. These specific kinases and phosphatases are
tightly regulated themselves.

In eukaryotes, phosphorlyation is used to control the activity of hundreds of enzymes. These enzymes are
phosphorylated in response to extracellular signals such as hormones or growth factors.
The addition of an adenylyl (AMP) group to a tyrosine is
another form of reversible covalent modification.
Adenylylation of a tyrosine is used to regulate glutamine
synthase, a key enzyme in nitrogen metabolism. The
tyrosine that is adenylylated is near the active site. The
addition of the bulky AMP group obstructs the active site
and inhibits the enzyme.
There are other groups that are covalently attached to
enzymes to regulate activity. These groups include fatty
acids, ioprenoid alcohols and carbohydrates.
Plants extensively use the reversible reduction of cystine
disulfide bridges to control enzyme activity. Many of the
enzymes involved in the breakdown of carbohydrates are
controlled this way.
III. Allosteric Regulation
All organisms use allosteric regulation in responding to
changes in conditions within a cell. Many enzymes have a
regulatory site which binds effector molecules. The
binding of an allosteric effector at this site causes a
conformational change in the enzyme which can either activate or inactivate the enzyme. The allosteric
effector that binds to the regulatory site does not need to have any structural similarity to the substrate.
Consider the metabolic pathway of histidine biosynthesis. The pathway consists of nine enzymes that
work one after another. Histidine biosynthesis is energetically expensive. If histidine is present in
abundance then it is advantageous to shut this pathway off. The first step is the phosphorylation of
phosphoribosylpyrophosphate by ATP. Neither of these substrates have any similarity to the final product
of the pathway, histidine, yet histidine binds to this enzyme at an allosteric binding site causing a
conformational change that inactivates the enzyme. Inhibition of the first step of metabolic pathways is a
common them in metabolism.
We have already seen allosteric interactions in the binding of oxygen by hemoglobin. The cooperativity
of hemoglobin results for quaternary structure interactions
between the four subunits. Most enzymes that are allosterically
regulated have multiple subunits and the conformational changes
that produce changes in activity are often related to interacting
subunits. We are now familiar with Michealis-Menten Kinetics
which produce hyperbolic substrate-velocity curves similar to the
oxygen binding by myoglobin. Many enzymes that are
allosterically regulated do not obey the classical kinetic equation,
rather they are sigmoidal like hemoglobin.
The kinetics of the reaction catalyzed by phosphofructokinase.
In the presence of 1.5 mM ATP the rate has a sigmoidal

dependence on the concentration of the substrate F-6-P. The sigmoidal shape is due to ATP binding to an
inhibitory site on the enzyme. This inhibition is overcome by the addition of AMP. ATP binding to PFK
is an example of negative cooperativity.
This is reflected by the hill equation which we have derived for hemoglobin, but extends easily to
enzymes. Ys is the fraction of enzyme with substrate bound. K is the product of the individual
dissociation constants. n is the Hill coefficient, a measure of cooperativity. If n=1, there is no
cooperativity, if n>1 positive cooperativity, if n<1 then negative cooperativity. The equation was derived
under the infinite cooperativity assumption which means the enzyme is either free or has substrate bound
at every site. Despite this over simplifying assumption, the hill coefficient provides a useful measure of
cooperativity.
2
[S]
[S]n
Ys =
Ys =
2
n
K1K2 + [S] .
K + [S]
For the two subunit case:
The linear form of the Hill equation is shown below:

Log

YS
1 - YS

= nlog[S] - logK

If the binding of the substrate is sequential rather than all or

none(the assumption of infinite cooperativity), then the slope will change as function of [S]. The hill
coefficient is defined to be the slope of the line when the enzyme is half saturated. When the enzyme is
half saturated Ys=1-Ys, the log (Ys/(1-Ys))=0. So the Hill coefficient is the slope of the line at the
intercept of the x-axis.
Enzymes with Allosteric Transitions that Resemble Hemoglobin.

There are many allosterically regulated enzymes in nature which occupy key positions in metabolism.
There are three other enzymes in addition to ATCase and hemoglobin which have crystal structures of the
R and T states: Phosphofructokinase, fructose-1,6-bisphosphatase, and glycogen phosphorylase. All five
of these proteins cooperatively bind ligands or substrates. The transitions from the RT states are
concerted and preserve the symmetry of the proteins. These proteins all have two sets of alternative
contacts which are stabilized by salt-bridges and hydrogen bonds. The quaternary shifts require every
subunit to move in relation to each other through rotations and translations which preserve the symmetry
of the whole molecule.
IV. Isozymes
Isozymes are enzymes that have different amino acid sequences but catalyze the same reaction. Isozymes
usually have different kinetic parameters, Km and Vmax, and different regulatory properties. Different
isozymes are found in different tissues or expressed during different developmental stages. The classic
example of isozymes is lactate dehydrogenase, LDH. LDH catalyzes the following reversible reaction:
NADH

H3C

NAD+ + H+

H3C

O-

OH

Pyruvate
NADH

NAD + H

Lactate Dehydrogenase

Lactate

O-

Human beings have 2 isozymes of this enzyme. One isozyme is dominant in the muscle tissue and
functions during anaerobic glycolysis converting pyruvate into lactate to regenerate NAD+. Excess lactate
is released by the exercising muscles into the blood stream. This isozyme of LDH is type M. The other
isozyme of LDH is predominant in the heart muscle, type H. Heart muscle always functions under
aerobic conditions. Heart muscle can utilize lactate as an energy source. Heart muscle absorbs lactate
from the blood stream where the H-LDH isozyme coverts lactate into pyruvate. Pyruvate is then used as a
fuel source. These two isozymes of LDH are 75% homologous. Both active LDH enzymes have a
tetrameric tertiary structure. Functional tetrameric LDH enzymes may have 5 different combinations of
the two isozyme polypeptides (LDH-1 to LDH-5). The highest concentrations of LDH-1 (H4 ) are found
in the heart muscle tissue, the highest levels of LDH-5 (M4 ) are found in the liver and skeletal muscle.
The M4 isozymes functions optimally in an anaerobic environment; the H4 isozyme functions optimally in
an aerobic environment. The proportion of M/H isozymes is altered during the course of development of
the heart as the tissue switches from the anaerobic environment of the uterus to the aerobic environment
of our atmosphere. Shown below is the change in heart LDH isozymes during the course of development.
The M isozyme is denoted by blue circles, the H isozyme is denoted by red squares. The numbers on the
horizontal axis denote days before and after birth.

Shown below is the tissue specific forms of LDH isozymes. The appearance of some isozymes in the
blood serum is indicative of tissue damage. For example, increase in the serum concentration of LDH-1
(H4 )is indicative of a heart attack. Increased serum levels of LDH-5 (M4) is indicative of liver or skeletal
muscle damage.