Regulatory strategies Attila Ambrus. aspartate trans- carbamoylase. 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.
first step in pyrimidine biosynthesis
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 homotropiceffect
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 agreater conformational change than for
induced fit and it is transmittedthrough the whole 3D structure; this
can promote activation or inhibitionfor the enzymatic function. Regula-
tory efficiency is dependent on the actual balance of the concentrations
of S and the allosteric ligand.
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)
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 bind-
ing 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).
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 coordina-
tion 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).
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
hydroxylation of Pro stabilizes collagen fibers (implication of scurvy)
lack ofg-carboxilation of Glu in prothrombin leads to hemorrhage in
Vitamin K deficiency
secreted or cell-surface proteins are often glucosylated on Asnfor being
more hydrophylic and able to interact with other proteins
addition of fatty acids to the NH2-terminus or Cys makes the protein more
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 (cova-
lently 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 ispossible viaproteolytic 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
4. Proteolytic activation: activation from proenzymes or zymogens (see
before; e.g. digestive Es like chymotrypsin, trypsin, pepsin). Blood coagula-
tion is a greatexample 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
Allostery at ATCase:
bigger, catalytic sub-
unit, unresponsive to
CTP, no sigmoidal kine-
tics,3 chains (34 kDa
smaller,regulatory subunit,binds CTP
no catalytic activity,2 chains (17 kDa
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 reaction is shut off
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.
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).
ternary structure upon binding I
(trimers move 12 Å apart, rotate 10o
dimers rotate 15o (T and R states))
from other subunits!
ce the purine and pyrimidine
nucleotide pools and signals
that the cell has energy for
mRNA synthesis and DNA
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), func-
tions 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
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 myo-
cardial infarction has damaged heart muscle cells leading to release of
cellular material (good for clinical diagnosis).
Acetyltransferases and deacetylases are themselves regulated by phospho-
rylation: covalent modification can be controlled by the covalent modifica-
tion of the modifying E.
Allosteric properties of many Es are modified by covalent modifications.
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 reactioncan 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 kina-
ses are usually intracellular proteins where the donor (ATP) is abundant.
Phosphatases generally turn off signaling pathways what kinases triggerred.
1. Adds 2 negative charges that may perturb/rearrange electrostatic
interactions inthe protein and alter S binding and activity.
2. A phosporyl group is able to form 3 or more (new) H-bonds that may
3. Itcan change theconformational equilibrium constant between diffe-
rent functional statesby 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 hydro-
phobic one (Lys can substitute for an Arg with some loss of affinity). Synt-
hetic peptides also react, so nearby aa seq. what determines specificity.
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.
Isoenzymesare typical for kinases to fine-tune regulation in specific cells
or developmental stages.
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 mo-
dification 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.
- activation leads to the formation of a S-binding site by triggering a con-
formational 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
cigarette smoking causes this reaction, and since Met 358 is
essential for binding elastase, inhibition and protection against
tissue damage weakens for smokers.