Transcript Slide 1

Lecture 16:
Regulation of Proteins 3:
Isozymes and
Covalent Modification
Isozymes
Covalent Modification
Protein Kinase A
Biological Processes are Carefully Regulated
Allosteric Control:
The activity of some proteins can be controlled by modulating
the levels of small signalling molecules. The binding of these
molecules causes conformational changes in the protein
which affect its activity.
Multiple forms of Enzymes:
Different tissues or developmental stages sometimes have specific
versions of a given enzyme which have distinct properties although
they may have the same basic activity.
Reversible Covalent Modification:
The activity of many proteins is controlled by attachment of small
chemical groups. The most common such modification is
phosphorylation- attachment of a phosphate group.
Proteolytic Activation:
Some enzymes are synthesized in an inactive form and must be
activated by cleavage of the inactive form.
Multiple Forms of Proteins
Different tissues or developmental stages differ in their requirements
for the activity of various proteins.
An example is provided by fetal hemoglobin. Oxygen is provided to
the fetus through the mother’s circulatory system, which requires
that oxygen be transferred from the maternal hemoglobin to the
hemoglobin in the bloodstream of the fetus. The fetus expresses
a different form of hemoglobin which has a higher oxygen affinity
than adult hemoglobin, facilitating this transfer.
This regulatory strategy is also a common means of controlling
the activity of enzymes.
Enzymes that carry out the same chemical reaction, but differ in
sequence, kinetic properties, and/or in regulatory properties,
are called isozymes.
Isozymes of Lactate Dehydrogenase
Lactate dehydrogenase (LDH) functions in glucose metabolism.
Mammals have 2 versions of LDH, the H isozyme (found in heart)
and the M isozyme (found in skeletal muscle) which are closely
related, sharing about 75% sequence identity.
LDH functions as a tetramer, but individual tetramers can be
composed of any combination of M and H isozymes.
(M4, M3H, M2H2, MH3, or H4)
The M4 tetramer functions optimally under anaerobic
conditions, while the H4 tetramer functions optimally under
aerobic conditions.
The H4 tetramer has higher substrate affinity and also can be
allosterically inhibited by pyruvate. The M4 tetramer has a lower
affinity for substrate and is not allosterically regulated.
The mixed tetramers exhibit properties intermediate between the
the H4 and M4 tetramers- differential expression of the two isozymes
allows fine control over the LDH activity.
Developmental Regulation of LDH Isozymes
At different developmental stages in the rat heart, the two isozymes of
LDH are expressed to different degrees. In the early embryo the M
form predominates, and in the adult the H form predominates.
H4
M4
(anaerobic)
Age (days)
(aerobic)
Tissue-Specific LDH Isozyme Expression
The levels of the different isozymes also vary from tissue to tissue.
There is a distribution of different complexes in a given tissue.
LDH-1
LDH-2
LDH-3
LDH-4
LDH-5
Disruption of cells can lead to release of LDH into the bloodstream.
In blood, the level of LDH-2 is normally higher than the level of LDH-1.
But during a heart attack, LDH-1 can be released into the bloodstream,
increasing it above the level of LDH-2. Analysis of the relative levels
of LDH isozymes serves as a diagnostic for a variety of medical
conditions.
Covalent Modification
The activity of many proteins is controlled by various forms of covalent
modification- attachment of a small functional group. The modification
can increase or suppress the activity of the proteins.
Specific enzymes carry out these modifications, and other enzymes
remove them.
In most cases the modification is reversible- removal of the attached
group reverses the effect of the modification.
In this way the regulated proteins can be “turned on” and “turned off”
as appropriate, depending on the cell’s need for that particular activity.
Examples of Covalent Modification
A variety of modifications exist, acting to control a wide range
of cellular functions.
Acetylation of Histones
Histones are important eukaryotic proteins involved in the packaging
of DNA in chromosomes and also in regulation of gene expression.
Histones
DNA
Section of
chromatin fiber
Histones are basic proteins, rich in Lys and Arg residues. The positive
charge assists in binding DNA, which is negatively charged.
The lysine residues of histones near regions of actively transcribed genes
are heavily acetylated.
Acetylation of lysines in histones removes the positive charge, weakening
the affinity of histones for DNA. This makes it easier to remove or displace
the histone from DNA to enable genes to be transcribed.
The covalent modfication of histones is carried out by enzymes called
histone acetyltransferases. Removal of the acetyl group is carried out
by deacetylases. These enzymes allow changes in the expression
level of genes in various locations on the chromosome.
In turn, the acetyltransferases and deacetylases are themselves regulated
by phosphorylation.
Phosphorylation
Probably the most widespread method of regulation is
phosphorylation- attachment of a phosphate group.
A wide variety of enzymes, many membrane channels and transcription
factors, and virtually all metabolic processes are regulated
by phosphorylation.
Phosphorylation is carried out by protein kinases, enzymes which
transfer a phosphate group from ATP to hydroxyl groups in proteins.
Kinases
A kinase is any enzyme which transfers a phosphate group. Kinases
are named for the target which receives the phosphate. (eg glycerol
kinase phosphorylates glycerol)
The phosphate group derives from ATP, which is only present inside
cells. Extracellular proteins are not regulated by phosphorylation.
An enzyme that phosphorylates a protein is a protein kinase.
Two major classes of protein kinases:
Serine/threonine kinases
Group that receives the phosphate is a serine or threonine hydroxyl.
Tyrosine kinases.
Group that receives the phosphate is a tyrosine hydroxyl.
Examples of Protein Kinases
Protein kinases comprise one of the largest protein families known- there
are more than 500 in humans. These allow regulation specific to particular
tissues, developmental stages, and substrate proteins.
Kinases themselves are controlled by many different kinds of cellular
signals including by other kinases.
Kinases and Phosphatases
Phosphorylation is carried out by kinases.
Removal of phosphate groups is carried out by phosphatases.
These reactions are not the reverse of one another.
Both reactions are energetically downhill and so are unidirectional
but proceed extremely slowly in the absence of enzymes.
Kinase
Phosphatase
Transfer of phosphate
from ATP
to protein.
Transfer of phosphate
from protein
to water.
Effects of Phosphorylation
Phosphorylation often activates a target molecule, for example by
inducing a conformational change to a more active state. The influence
on target activity can be accomplished through various effects.
Charge: Phosphorylation adds 2 negative charges which can
participate in (or disrupt) charge-charge interactions.
Hydrogen bonding: The phosphate group can participate in 3 or more
hydrogen bonds.
Energy: The substantial amount of energy in the phosphate bond
can strongly affect conformational equilibria.
Time: Phosphorylation can be accomplished in seconds and can last for
as long as needed. By regulating the phosphorylation and dephosphorylation
steps, the activity of the target can be adjusted to synchronize with a
physiological process.
Amplification: A single kinase can phosphorylate and activate hundreds of
target molecules resulting in a large effect from a small stimulus.
Specificity of Protein Kinases
Dedicated protein kinases phosphorylate only a single target or a few
closely related ones, allowing fine control over this limited target.
Multifunctional protein kinases phosphorylate many different targets,
allowing a single kinase to control a variety of different processes.
For kinases with many different targets, comparison of the amino acid
sequences of the residues near the phosphorylation sites identifies
patterns. The primary determinant of specificity is the amino acids
surrounding the phosphorylation site.
For example, protein kinase A recognizes and phosphorylates the serine or
threonine in the sequences:
Arg-Arg-X-Ser-Z or Arg-Arg-X-Thr-Z
where X is a small residue (eg Gly)
and Z is a large hydrophobic residue (eg Ile)
Protein Kinase A
Many physiological processes are regulated by hormones, which are
are extracellular signalling molecules.
Hormones bind to receptors at the cell surface. In some cases, the
binding of hormones causes the formation of intracellular signalling
molecules, or second messengers, such as cyclic AMP.
This carries the signal from the hormone into the cell and results in
activation of many different proteins. Most of these proteins are
activated by a single kinase, protein kinase A. (PKA)
Protein Kinase A is Regulated by cAMP
PKA consists of:
2 C subunits (catalytic subunits) that contain the kinase activity
2 R subunits (regulatory subunits) that bind cAMP
In the absence of cAMP, an inactive C2R2 complex is formed in which the
regulatory subunits tightly bind and sequester the catalytic subunits.
In the presence of cAMP (eg in response to a hormone) the regulatory
subunit binds the cAMP, inducing a conformational change that releases
the catalytic subunits, which can then begin phosphorylating their targets.
Mechanism of cAMP Regulation
The R subunit of PKA contains a sequence nearly matching the preferred
substrate of the C subunits but incapable of being phosphorylated.
This pseudo-substrate sequence contains the residues
…Arg-Arg-Gly-Ala-Ile…
and the C subunit binds this sequence tightly at its active site, preventing
other substrates from being phosphorylated.
The binding of cAMP by the R subunit causes an allosteric conformational
shift which removes the pseudo-substrate sequence from
the C subunit, releasing it.
Structural Basis of cAMP Regulation
The structure of the catalytic subunit of PKA in complex with a
pseudosubstrate peptide allows the substrate specificity and inhibition
by the R subunit to be understood as due to complementarity with
specific residues in the c subunit.
Arg-Arg-Asn-Ala-Ile
Ionic
interactions
Catalytic subunit
Hydrophobic interactions
Summary:
Isozymes are enzymes which have the same activity but different kinetics
or regulatory properties- differential expression of isozymes allows control
over enzyme activity.
Many proteins are regulated by covalent modification. The most common
such modification is phosphorylation.
Protein kinase A carries out phosphorylation of a wide variety of
targets in response to cyclic AMP.
Key Concepts:
Isozymes of LDH
Regulation of histones by acetylation
Kinases
Phosphatases
Role of Protein Kinase A