Biochemistry 6/e

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Regulatory strategies Attila Ambrus
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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 Es 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.
Regula- tory 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)
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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 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).
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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 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).
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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
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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 is possible via proteolytic
cleavage (not to be mixed up with preproteins
preproteinproteinsignal peptide many times
first a pre-proprotein is synthesized that is
cleaved then to the proprotein)
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4. Proteolytic activation activation from
proenzymes or zymogens (see before e.g.
digestive Es like chymotrypsin, trypsin, pepsin).
Blood coagula- tion 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
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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 by CTP.
CTP has very small structural similarity to the
Es 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.
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• The dissociated subunits can easily be separated
  based on their great
• difference in charge (by ion-exchange
  chromatography) or size (by suc-
• rose 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
• ATCase is composed of discrete subunits
• solely the physical interaction amongst subunits
  secures allostery

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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).
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1 AS/subunit, great change in qua- ternary
structure upon binding I (trimers move 12 Å
apart, rotate 10o dimers rotate 15o (T and R
states))
from other subunits!
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concerted mechanism
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high ATP levels try to balan- ce the purine and
pyrimidine nucleotide pools and signals that the
cell has energy for mRNA synthesis and DNA
replication
T
R
LT/R
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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),
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 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 myo- cardial infarction has
damaged heart muscle cells leading to release of
cellular material (good for clinical diagnosis).
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Covalent modifications
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.
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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
gt500 (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 kina- ses are usually
intracellular proteins where the donor (ATP) is
abundant. Phosphatases generally turn off
signaling pathways what kinases triggerred.
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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
diffe- rent 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 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.
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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. PKAs
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.
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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
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. Examples
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• 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. pro-
• insulin insulin)
• - collagen, the major component of skin and bone,
  is derived from a pro-
• collagen 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).

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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 con- formational
change (revealed by the 3D structures
determined) - the newly formed Ile N-terminuss
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

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Trypsinogen activation

• much greater structural changes (15 of aas)
  than in case of chymo-
• trypsin
• 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 side-
• chain cleavage preferences requires a common
  activator of pancreatic zy-
• mogens 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

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• 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 CO 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 panc-
• reatic ducts to prevent premature activation of
  trypsin and zymogens (that
• would cause tissue damage and acute pancreatitis)
• - there is also a1-antitrypsin (a1-antiproteinase)
  , 53 kDa, in plasma, pro-
• tects tissues from elastase secreted by
  neutrophils (there are genetic
• disorders where digestion of tissues occurs)

cigarette smoking causes this reaction, and since
Met 358 is essential for binding elastase,
inhibition and protection against tissue damage
weakens for smokers.