Regulatory strategies
Attila Ambrus
aspartate transcarbamoylase
first step in pyrimidine biosynthesis
Es often must be regulated so that they function only at the right
place and time.
Regulation is essential for coordinating the complexity of biochemical
processes in an organism.
E activity is regulated in five principal ways:
1. Allosterically: Heterotropic or homotropic effect
Heterotropic: a small signal molecule reversibly binds to the E’s regulatory
site (which is usually far from the AS); the signal molecule has a different
structure than S has. There is a greater conformational change than for
induced fit and it is transmitted through the whole 3D structure; this
can promote activation or inhibition for the enzymatic function. Regulatory efficiency is dependent on the actual balance of the concentrations
of S and the allosteric ligand.
Activators may:
i. increase the affinity of E towards S; KM decreases
ii. provide better orientation for catalytic aas; Vmax increases
iii. induce the active conformation (w/o ligand, no E activity at all)
Inhibitors may:
i. induce inactive conformation (here often S binding induces a
conformation that does not let the allosteric inhibitor bind; kinetic
picture: apparent competitive inhibition)
ii. decreases catalytic velocity via the induced conformational change;
kinetic picture: apparent non-competitive inhibition)
Homotropic: in protein complexes of oligomeric nature consisiting of
identical subunits. Here the allosteric ligand is the S itself (for the other
subunits the conformations of which are also changing just by binding S to
one of the subunits). This cooperativity in action enhances substrate binding efficacy at the other binding sites, results in non-M-M kinetics and
a sigmoidal S saturation curve. True mechanism is still under investigation,
but we have two models to describe the effects: symmetry and sequential
models (see in details in the Hb/Mb lecture). The homotropic effect
provides a much tighter control over S binding and release and may happen
also for proteins having no enzymatic activities or Es having multiple
binding sites for S in a single polypeptide chain.
The first step of a metabolic pathway is generally an allosteric E. This E
has control over the necessity of starting or stopping a pathway. The last
P of the pathway generally allosterically inhibits this E (feedback inhibition).
In other instances the abundant amount of the material to be converted
activates this E (precursor activation). There are also examples that the
same molecule is an allosteric activator and an inhibitor in the same time,
for the same pathway, but of its reverse directions (giving tight coordination for the directionality of the metabolic processes).
The allosteric affect can be defined more generally: all conformational/
functional changes caused by ligand binding (to a site other than the AS)
can be considered an allosteric effect.
E.g. ligand binding alters protein-protein (like for hormone-receptor action)
or protein-DNA (like for transcription control in prokaryotes) interactions.
These kind of regulatory controls are so general in biochemistry that we
sometimes do not even mention that it is actually an allosteric action.
2. Isoenzymes: It is possible by them to vary regulation of the same
reaction at different places and metabolic status in the same organism.
Isoenzymes are homologous Es in the same organism catalyzing the same
reaction but differ slightly in structure, regulatory properties, KM or Vmax.
Often isoenzymes get expressed to fine-tune the needs of metabolism in
distinct tissues/organelles or developmental stages. They get expressed
from different genes (by gene duplication and divergence).
3. Covalent modification: catalytic and other properties of enzymes (and
proteins in general) get often markedly altered by a covalent modification
E.g. phosporylation at Ser,Thr or Tyr by protein kinases (using ATP as
phosphoryl donor, triggered generally by hormon or growth factor action);
dephosphorylation takes place by phosphatases (implications in signal
transduction and regulation of metabolism)
other important covalent modifications:
acetylation of NH2-terminus makes proteins more stable against
degradation
hydroxylation of Pro stabilizes collagen fibers (implication of scurvy)
lack of g-carboxilation of Glu in prothrombin leads to hemorrhage in
Vitamin K deficiency
secreted or cell-surface proteins are often glucosylated on Asn for being
more hydrophylic and able to interact with other proteins
addition of fatty acids to the NH2-terminus or Cys makes the protein more
hydrophobic
no new adduct, but a spontaneous rearrangement (and oxidation) of a
tripeptide (Ser-Tyr-Gly) inside the protein occurs in green fluorescent
protein (GFP, produced by certain jellyfish) that results in fluorescence
(great tool as a marker in research)
fluorescence micrograph of a 4-cell C.elegans
embryo in which a PIE-1 protein labeled (covalently linked) with GFP is selectively emerges
in only one of the cells (cells are outlined)
some proteins are synthesized as inactive precursors (proprotein, zymogen)
and stored until use; activation is possible via proteolytic cleavage
(not to be mixed up with preproteins; preprotein=protein+signal peptide;
many times first a pre-proprotein is synthesized that is cleaved then to the
proprotein)
4. Proteolytic activation: activation from proenzymes or zymogens (see
before; e.g. digestive Es like chymotrypsin, trypsin, pepsin). Blood coagulation is a great example for a cascade of zymogen activations. Many of
these Es cycle between inactive and active forms. Generally there is an
irreversible activation by hydrolysis of sometimes even one specific bond
yielding the active form of E. The digestive and clotting Es can then be
shut off by irreversible binding of inhibitory proteins.
5. Controlling enzyme amount: this takes place most often at the level of
transcriptional regulation
Allostery at ATCase:
How to regulate the amount of CTP needed for the cell?
It was found that CTP in a feedback inhibition acts on the ATCase reaction.
If there is too much (enough) of CTP, simply ATCase
is shut off
treatedreaction
with Hg-compound
native E
by CTP.
(11.6S)
(5.8S)
(2.8S)
bigger, catalytic subunit, unresponsive to
CTP, no sigmoidal kinetics,3 chains (34 kDa
each)
smaller,regulatory subunit,binds CTP
no catalytic activity,2 chains (17 kDa
each)
ultracentrifugation
CTP
has very small structural similarity to the E’s S or P, hence it needs
to bind to a regulatory (allosteric site). CTP is an allosteric inhibitor, that
actually binds to another polypeptide chain than where the AS is.
ATCase has separable regulatory and catalytic subunits.
4 Cys subunits
(where Hg can
The dissociated
canact!)
easily be separated based on their great
difference in charge (by ion-exchange chromatography) or size (by sucrose density gradient centrifugation). The Hg-derivative can be eliminated
by b-SH-EtOH.
If the subunits are mixed again, they form the original E complex again
with 2 catalytic trimers and 3 regulatory dimers.
2c3 + 3r2 = c6r6
Most strikingly, the reconstituted E shows the same allosteric and kinetic
properties as the native E.
This means that:
1. ATCase is composed of discrete subunits
2. solely the physical interaction amongst subunits secures allostery
They found the AS by crystallizing the E with a bi-S-analog (analog of the
2 Ss) that resembles a catalytic intermedier (competitive inhibitor).
1 AS/subunit, great change in quaternary structure upon binding I
(trimers move 12 Å apart, rotate 10o
dimers rotate 15o (T and R states))
from other subunits!
concerted mechanism
high ATP levels try to balance the purine and pyrimidine
nucleotide pools and signals
that the cell has energy for
mRNA synthesis and DNA
replication
R
T
L=[T]/[R]
Isoenzymes
They can be distinguished generally by their electrophoretic mobilities.
Example: Lactate dehydrogenase (LDH): humans have 2 major isoenzymes
of LDH, the H form (heart muscle) and the M form (skeletal muscle; AA
seq. is 75% the same). The functional E is a tetramer, and H and M can be
mixed in them.
H4: higher affinity for S, pyruvate allosterically inhibits it (not M4), functions optimally in the aerobic heart muscle
M4: functions optimally in the anaerobic condition of the skeletal muscle
Various combinations of the tetramer gives intermediate properties (see
Ch 16).
It is impressive how rat heart switches subunit composition as it develops
towards the H (square label) form. Also the tissue distribution of the LDH
isoenzymes can be seen on the other figure in adult rats.
Increase of H4 over H3M in human blood serum may indicate that myocardial infarction has damaged heart muscle cells leading to release of
cellular material (good for clinical diagnosis).
Covalent modifications
Acetyltransferases and deacetylases are themselves regulated by phosphorylation: covalent modification can be controlled by the covalent modification of the modifying E.
Allosteric properties of many Es are modified by covalent modifications.
Phosporylation-dephosporylation
30% of eukaryotic proteins are phosphorylated. It is virtually everywhere
in the body regulating various sorts of metabolic processes and pathways.
Phosphorylation is carried out by protein kinases whilst dephosphorylation
is performed by protein phosphatases. These constitute one of the largest
E families known: >500 (homologous) kinases in humans. This means that the
same reaction can really be fine-tuned to tissues, time, Ss.
Most commonly ATP is the phosphoryl donor (the terminal (g) phosphoryl
group is transferred to a specific aa). One class of kinases handles Ser
and Thr transfers, another class does Tyr ones (Tyr kinases are unique in
multicellular organisms, principally important in growth regulation, and
mutants often show up in cancers).
Extracellular Es are generally not regulated by phosporylation; Ss of kinases are usually intracellular proteins where the donor (ATP) is abundant.
Phosphatases generally turn off signaling pathways what kinases triggerred.
Reasons why phophorylation(/dephosphorylation) may be effective on
protein structures:
1. Adds 2 negative charges that may perturb/rearrange electrostatic
interactions in the protein and alter S binding and activity.
2. A phosporyl group is able to form 3 or more (new) H-bonds that may
alter structure.
3. It can change the conformational equilibrium constant between different functional states by the order of 104.
4. It can evoke highly amplified effects: a single activated kinase can
phosphorylate hundreds of target proteins in short time. If the target
proteins are Es, they in turn can convert a great number of S molecules.
5. ATP is a cellular energy currency. Using this molecule as a phosphoryl
donor links the energy status of the cell to the regulation of metabolism.
Kinases vary in specificity: dedicated and multifunctional kinases. Protein
kinase A is from the latter type and recognizes the following consensus
sequence: Arg-Arg-X-Ser/Thr-Z, where X is a small aa, Z is a large hydrophobic one (Lys can substitute for an Arg with some loss of affinity). Synthetic peptides also react, so nearby aa seq. what determines specificity.
cAMP activates protein kinase A (PKA) by altering quaternary structure
Adrenaline (hormone, neurotransmitter) triggers the generation of cAMP,
an intracellular messenger, that then activates PKA. The kinase alters then
the function of several proteins by Ser/Thr-phosphorylation.
cAMP activates PKA allosterically at 10 nM (activation mechanism is similar
to the one in ATCase: C and R subunits).
If no cAMP: inactive R2C2; R contains: Arg-Arg-Gly-Ala-Ile (pseudo-S-seq.
that occupies the AS of C in R2C2, preventing the binding of real Ss).
Binding 2 cAMPs to each R: dissociation to R2 and 2 active Cs. cAMP binding
relieves inhibition by allosterically moving the pseudo-S out of the AS of C.
PKA’s aas 40-280 is a conserved catalytic core for almost all known kinases.
Isoenzymes are typical for kinases to fine-tune regulation in specific cells
or developmental stages.
Activation by specific proteolytic cleavage
Since ATP is not needed for this type of activation, Es outside the cell can
also be regulated this way.
This action, in contrast to molecules regulated by reversible covalent modification or allosteric control, happens once in the lifespan of a molecule
(completely irreversible modification). It is (generally) a very specific
cleavage that makes the target pro-E active.
Examples:
- blood clotting cascade of proteolytic activations makes the response to
trauma rapid (see Hemostasis lecture)
- some protein hormons are also zymogens when first synthesized (e.g. proinsulin – insulin)
- collagen, the major component of skin and bone, is derived from a procollagen precursor
- many developmental processes use active proteolysis: great amount of
collagen is degraded in the uterus after delivery (procollagenase turns to
collagenase in a timely fashion)
-Programmed cell death, or apoptosis, is mediated by proteases called
caspases generated from procaspases. Responding to certain signals (see
Apoptosis lecture, next semester), caspases cause cell death throughout
most of the animal kingdom (apoptosis gets rid of damaged or infected
cells and also sculpts the shapes of body parts during development).
Chymotrypsinogen activation
- chymotrypsin is a digestive enzyme that hydrolyses proteins in the small
intestine
- it is synthesized as inactive zymogen (chymotrypsinogen) in the pancreas
- activation is carried out by the specific cleavage of a single peptide bond
(Arg 15-Ile 16)
- activation leads to the formation of a S-binding site by triggering a conformational change (revealed by the 3D structures determined)
- the newly formed Ile N-terminus’s NH2-group turns inward and forms an
ionpair with Asp 194 in the interior of the E; this interaction triggers
further changes in conformation that ultimately create the S1 site:
Met 192 moves from a deeply buried position to the surface of the E and
residues 187 and 193 get more extended
-the correct position of one of the N-Hs in the oxyanion hole is also taken
only after the above conformational changes occured
Trypsinogen activation
- much greater structural changes (~15% of aas) than in case of chymotrypsin
- the four stretches, suffering the greatest changes, are quite flexible in
the zymogen while pretty structured in the mature E
- the oxyanion hole in the zymogen is too far from His 57 to promote the
tetrahedral intermedier
- the concurrent need in the duodenum for proteases with different sidechain cleavage preferences requires a common activator of pancreatic zymogens; this is trypsin.
- trypsin is generated from trypsinogen by enteropeptidase that hydrolyzes
a Lys-Ile peptide bond in trypsinogen; small amount of trypsin is enough to
speed up the auto-activation
- proteolytic activation can only be controlled by specific inhibitors; for
trypsin there exists a pancreatic trypsin inhibitor, 6 kDa, binding very
tightly to the AS (Kd=10-13 M; 8 M urea/6 M Gu-HCl cannot take them apart)
- the trypsin inhibitor is a very good S analog; X-ray studies show that the
I lies in the AS (Lys 15 of the I interacts with the Asp in the S1 pocket,
many H-bonds exist between the main chains of E and I, the C=O and
surrounding atoms of Lys 15 of I fit snugly to the AS)
- the structure of I is essentially unchanged upon binding to E, it is already
very complementary to the AS
- the Lys 15-Ala 16 bond is eventually cleaved, but very slowly: the t1/2 of
E-I is several months
- the I is practically a S, too complementary to AS, binds too tightly and
turns over very slowly
- small amount of such I exists; it works in the pancreas and the pancreatic ducts to prevent premature activation of trypsin and zymogens (that
would cause tissue damage and acute pancreatitis)
cigarette smoking causes this reaction, and since Met 358 is
essential for binding elastase, inhibition and protection against
also
a1-antitrypsin
(afor
53 kDa, in plasma,
tissue
damage weakens
smokers.
1-antiproteinase),
- there is
protects tissues from elastase secreted by neutrophils (there are genetic
disorders where digestion of tissues occurs)