Download REGULATORY ENZYMES

REGULATION OF ENZYME
ACTIVITY
Medical Biochemistry, Lecture 25
Lecture 25, Outline
• General properties of enzyme regulation
• Regulation of enzyme concentrations
• Allosteric enzymes and feedback inhibition
• Other effectors of catalytic activity
Metabolic Homeostasis
General Properties: Regulatory
Enzymes
• The biochemical pathways that you will soon be
studying are composed of groups of
coordinated enzymes that perform a specific
metabolic process. In general, these enzyme
groups are composed of many enzymes, only a
few of which are regulated by the mechanisms
described in this lecture. Regulatory enzymes
are usually the enzymes that are the ratelimiting, or committed step, in a pathway,
meaning that after this step a particular reaction
General Properties: Regulatory
Enzymes (cont)
• Frequently, regulatory enzymes are at or
near the initial steps in a pathway, or part of
a branch point or cross-over point between
pathways (where a metabolite can be
potentially converted into several products in
different pathways). In general, a cell needs
to conserve energy - therefore costly (in
metabolic terms) biosynthetic reaction
pathways will not be operational unless a
particular metabolite is required at a given
General Properties: Regulatory
Enzymes (cont)
• Recall that when acting as catalysts, enzyme
mediated-reactions should be reversible.
However, regulatory enzymes frequently
catalyze thermodynamically irreversible
reactions, that is, a large negative free energy
change (-∆G) greatly favors formation of a
given metabolic product rather than the reverse
reaction. Thus, regulation of enzyme activity,
usually at the committed step of the pathway, is
critical for supplying and maintaining cellular
metabolitic and energy homeostasis.
Two General Mechanisms that
Affect Enzyme Activity:
• 1) control of the overall
quantities of enzyme or
concentration of substrates
present
• 2) alteration of the catalytic
efficiency of the enzyme
Regulation of Enzyme
Concentrations
• The overall synthesis and degradation
of a particular enzyme, also termed its
turnover number, is one way of
regulating the quantity of an enzyme.
The amount of an enzyme in a cell can
be increased by increasing its rate of
synthesis, decreasing the rate of its
degradation, or both.
Regulation of Enzyme
Concentrations: Induction
• Induction (an increase caused by an effector
molecule) of enzyme synthesis is a common
mechanism - this can manifest itself at the level
of gene expression, RNA translation, and posttranslational modifications. The actions of
many hormones and/or growth factors on cells
will ultimately lead to an increase in the
expression and translation of "new" enzymes
not present prior to the signal. These
generalizations will be covered in more detail in
Dr. Bannon's lectures.
Regulation of Enzyme
Concentrations: Degradation
• The degradation of proteins is constantly
occuring in the cell, yet the molecular
mechanisms that determine when and
which enzymes will be degraded are poorly
understood. The turnover number of an
enzyme can be used for general
comparison with other enzymes or other
enzyme systems, yet these numbers can
vary from minutes to hours to days for
different enzymes.
Regulation of Enzyme
Concentrations: Degradation (cont)
• Protein degradation by proteases is
compartmentalized in the cell in the lysosome
(which is generally non-specific), or in
macromolecular complexes termed proteasomes.
Degradation by proteasomes is regulated by a
complex pathway involving transfer of a 76 aa
polypeptide, ubiquitin, to targeted proteins.
Ubiquination of protein targets it for degradation
by the proteasome. This pathway is highly
conserved in eukaryotes, but still poorly
understood
Regulation of Enzyme
Concentrations: Degradation (cont)
• Proteolytic degradation is an irreversible
mechanism. For examples, rapid proteolytic
degradation of enzymes that were activated in
response to some stimulus (for example, in a
signal transduction response). This type of
down-regulation allows for a transient response
to a stimulus instead of a continual response.
Establishing the links between proteasomes,
ubiquination and signal transduction pathways
is currently a very active research area
Zymogens: Inactive Precursor
Proteins
• A clinically important mechanism of controlling
enzyme activity is the case of protease
enzymes involved (predominantly) in food
digestion and blood clotting. Protease
enzymes (enzymes that degrade proteins) like
pepsin, trypsin and chymotrypsin are
synthesized first as larger, inactive precursor
proteins termed zymogens (specifically
pepsinogen, trypsinogen, and
chymotrypsinogen, respectively).
Zymogen Protease Examples
Chymotrypsinogen
cleavage sites to
yield active
chymotrypsin
Zymogens (cont)
• Activation of zymogens by proteolytic
cleavage result in irreversible activation.
Zymogen forms allow proteins to be
transported or stored in inactive forms that
can be readily converted to active forms in
response to some type of cellular signal.
Thus they represent a mechanism whereby
the levels of an enzyme/protein can be
rapidly increased (post-translationally). Other
examples of zymogens include proinsulin,
procollagen and many blood clotting
Enzyme/Substrate Compartmentation
• Segregation of metabolic processes into distinct
subcellular locations like the cytosol or
specialized organelles (nucleus, endoplasmic
reticulum, Golgi apparatus, lysosomes,
mitochondria, etc.) is another form of regulation.
Enzymes associated with a given pathway
frequently form organized, multi-component
macromolecular complexes that perform a
particular cellular process. Similarly, it follows
that the substrates associated with a given
pathway can also be localized to the same
organelle or cytosolic location. This segregation
Enzyme Regulation by
Compartmentation
Allosteric Enzymes
• Allosteric enzymes - from the Greek allos for
"other" and stereos for "shape" (or site)
meaning "other site". These enzymes function
through reversible, non-covalent binding of a
regulatory metabolite at a site other than the
catalytic, active site. When bound, these
metabolites do not participate in catalysis
directly, but lead to conformational changes in
one part of an enzyme that then affect the
overall conformation of the active site (causing
an increase or decrease in activity, hence these
metabolites are termed allosteric activators or
Allosteric Example
• Feedback Inhibition - This occurs
when an end-product of a pathway
accumulates as the metabolic demand
for it declines. This end-product in turn
binds to the regulatory enzyme at the
start of the pathway and decreases its
activity - the greater the end-product
levels the greater the inhibition of
enzyme activity. This can either effect
the Km or Vmax of the enzyme reaction.
Metabolic Pathway Product/
Feedback Inhibition
Allosteric Enzymes - Properties
• Allosteric enzymes differ from other
enzymes in that they are generally
larger in mass and are composed of
multiple subunits containing active sites
and regulatory molecule binding sites.
The same principles that govern binding
of a substrate to an active site are
similar for an allosteric regulator
molecule binding to its regulatory site.
Kinetics of Allosteric Enzymes - Terms
• Cooperativity - in relation to multiple subunit
enzymes, changes in the conformation of one subunit
leads to conformational changes in adjacent subunits.
These changes occur at the tertiary and quaternary
levels of protein organization and can be caused by an
allosteric regulator.
• Homotropic regulation - when binding of one
molecule to a multi-subunit enzyme causes a
conformational shift that affects the binding of the
same molecule to another subunit of the enzyme.
• Heterotropic regulation - when binding of one
molecule to a multi-subunit enzyme affects the binding
of a different molecule to this enzyme (Note: These
Allosteric Enzymes - Kinetics
• Allosteric enzymes do exhibit saturation kinetics at
high [S], but they have a characteristic sigmoidal
saturation curve rather than hyperbolic curve when vo
is plotted versus [S] (analogous to the oxygen
saturation curves of myoglobin vs. hemoglobin).
Addition of an allosteric activator (+) tends to shift the
curve to a more hyperbolic profile (more like
Michaelis-Menten curves), while an allosteric inhibitor
(-) will result in more pronounced sigmoidal curves.
The sigmoidicity is thought to result from the
cooperativity of structural changes between enzyme
subunits (again similar to oxygen binding to
hemoglobin). NOTE: A true Km cannot be determined
Vo vs [S] for Allosteric Enzymes
Models of Allosteric Proteins
Regulation by Modulator
Proteins - Calmodulin
Calmodulin is a small protein
(17 kDa) that can bind up to four
calcium ions (blue dots) in the
two globular domains. When
calciumis bound, calmodulin acts
as a protein co-factor to stimulate
the activity of target regulatory
kinases like phosphorylase kinase,
myosin kinase, Ca-ATPase and a
Ca/calmodulin-dependent
protein kinase. It is the structural
conformation of Ca-calmodulin
that makes it an active co-factor
Regulation of Enzyme Activity
by Covalent Modifications
• Another common regulatory mechanism is the
reversible covalent modification of an enzyme.
Phosphorylation, whereby a phosphate is
transferred from an activated donor (usually
ATP) to an amino acid on the regulatory enyme,
is the most common example of this type of
regulation. Frequently this phosphorylation
occurs in response to some stimulus (like a
hormone or growth factor) that will either
activate or inactivate target enzymes via
Phosphorylation/Signal Transduction
• Phosphorylation of one enzyme can lead to
phosphorylation of a different enzyme which in
turn acts on another enzyme, and so on. An
example of this type of phosphorylation
cascade is the response of a cell to cyclic AMP
and its effect on glycogen metabolism. Use of
a phosphorylation cascade allows a cell to
respond to a signal at the cell surface and
transmit the effects of that signal to intracellular
enzymes (usually within the cytosol and
nucleus) that modify a cellular process. This
process is generically referred to as being part
Signaling Regulation of Glycogen
Synthase and Phosphorylase
A-forms, most active
B-forms, less active
Other covalent modificiations:
• Prenylation, Myristoylation, Palmitoylation:
The covalent addition of hydrophobic, acyl
fatty acid or isoprenoid groups to soluble
proteins/enzymes can alter their intracellular
location. This type of hydrophobic acylation
generally causes target proteins to associate
with a membrane rather than the cytosol.
Thus, it represents a mechanistic and
functional re-compartmentalization of the
target protein/enzyme (an example of a
prenylated protein is the Ras oncogene
discussed in lecture 11)
Allosteric and Phosphorylation
Regulation - Glycogen Phosphorylase