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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.

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Regulatory strategies Attila Ambrus

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Regulatory strategies attila ambrus

Regulatory strategies

Attila Ambrus


Regulatory strategies attila ambrus

aspartate trans-

carbamoylase

first step in pyrimidine biosynthesis


Regulatory strategies attila ambrus

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 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.

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)


Regulatory strategies attila ambrus

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).


Regulatory strategies attila ambrus

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).


Regulatory strategies attila ambrus

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 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

hydrophobic


Regulatory strategies attila ambrus

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

proprotein)


Regulatory strategies attila ambrus

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

transcriptional regulation

Allostery at ATCase:


Regulatory strategies attila ambrus

treated with Hg-compound

native E

(11.6S)

(5.8S)

(2.8S)

bigger, catalytic sub-

unit, unresponsive to

CTP, no sigmoidal kine-

tics,3 chains (34 kDa

each)

smaller,regulatory subunit,binds CTP

no catalytic activity,2 chains (17 kDa

each)

ultracentrifugation

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 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.


Regulatory strategies attila ambrus

4 Cys (where Hg can act!)

  • The dissociated subunitscan 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 discretesubunits

  • solely the physical interaction amongst subunits secures allostery


Regulatory strategies attila ambrus

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).


Regulatory strategies attila ambrus

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!


Regulatory strategies attila ambrus

concerted mechanism


Regulatory strategies attila ambrus

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

L=[T]/[R]


Regulatory strategies attila ambrus

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).


Regulatory strategies attila ambrus

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.


Regulatory strategies attila ambrus

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 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.


Regulatory strategies attila ambrus

Reasons why phophorylation(/dephosphorylation) may be effective on

protein structures:

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

alter structure.

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.


Regulatory strategies attila ambrus

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.

Isoenzymesare typical for kinases to fine-tune regulation in specific cells

or developmental stages.


Regulatory strategies attila ambrus

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:


Regulatory strategies attila ambrus

  • 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).


Regulatory strategies attila ambrus

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 thespecific 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-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

  • thecorrect position of one of the N-Hs in the oxyanion holeis also taken

  • only after the above conformational changes occured


Regulatory strategies attila ambrus

Trypsinogen activation

  • much greater structural changes (~15% of aas) than in case of chymo-

  • trypsin

  • thefour stretches, suffering the greatest changes, are quite flexible in

  • the zymogen while pretty structured in the mature E

  • theoxyanion holein the zymogenis too far from His 57to 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

  • tightly to the AS (Kd=10-13 M; 8 M urea/6 M Gu-HCl cannot take them apart)


Regulatory strategies attila ambrus

  • 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 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.


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