Regulatory strategies

Attila Ambrus

aspartate trans-carbamoylase

first step in pyrimidine biosynthesis

Es often must be regulated so that they function only at the rightplace 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 regulatorysite (which is usually far from the AS); the signal molecule has a differentstructure than S has. There is a greater conformational change than forinduced 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 concentrationsof 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 increasesiii. 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 othersubunits the conformations of which are also changing just by binding S toone of the subunits). This cooperativity in action enhances substrate bind-ing efficacy at the other binding sites, results in non-M-M kinetics anda sigmoidal S saturation curve. True mechanism is still under investigation,but we have two models to describe the effects: symmetry and sequentialmodels (see in details in the Hb/Mb lecture). The homotropic effect provides a much tighter control over S binding and release and may happenalso for proteins having no enzymatic activities or Es having multiplebinding sites for S in a single polypeptide chain.

The first step of a metabolic pathway is generally an allosteric E. This Ehas 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 thesame 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 samereaction 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-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)

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)

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)

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 anirreversible activation by hydrolysis of sometimes even one specific bondyielding 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 reaction is shut off by CTP.

CTP has very small structural similarity to the E’s S or P, hence it needsto bind to a regulatory (allosteric site). CTP is an allosteric inhibitor, thatactually binds to another polypeptide chain than where the AS is.

ATCase has separable regulatory and catalytic subunits.

ultracentrifugation

native E treated with Hg-compound(11.6S)

(2.8S)

(5.8S)

bigger, catalytic sub-unit, unresponsive toCTP, no sigmoidal kine-tics,3 chains (34 kDa each)

smaller,regulatory subunit,binds CTPno catalytic activity,2 chains (17 kDa each)

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 -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 kineticproperties as the native E.

This means that:

1. ATCase is composed of discrete subunits2. solely the physical interaction amongst subunits secures allostery

4 Cys (where Hg can act!)

They found the AS by crystallizing the E with a bi-S-analog (analog of the2 Ss) that resembles a catalytic intermedier (competitive inhibitor).

from other subunits!

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))

concerted mechanism

high ATP levels try to balan-ce the purine and pyrimidinenucleotide pools and signalsthat the cell has energy formRNA synthesis and DNA replication

R T

L=[T]/[R]

IsoenzymesThey can be distinguished generally by their electrophoretic mobilities.

Example: Lactate dehydrogenase (LDH): humans have 2 major isoenzymesof 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 bemixed 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 LDHisoenzymes 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).

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.

Phosporylation-dephosporylation

30% of eukaryotic proteins are phosphorylated. It is virtually everywherein the body regulating various sorts of metabolic processes and pathways.

Phosphorylation is carried out by protein kinases whilst dephosphorylationis performed by protein phosphatases. These constitute one of the largestE families known: >500 (homologous) kinases in humans. This means that thesame reaction can really be fine-tuned to tissues, time, Ss.

Most commonly ATP is the phosphoryl donor (the terminal () phosphorylgroup is transferred to a specific aa). One class of kinases handles Serand 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.

Reasons why phophorylation(/dephosphorylation) may be effective on protein structures:

1. Adds 2 negative charges that may perturb/rearrange electrostaticinteractions in the protein and alter S binding and activity.

2. A phosporyl group is able to form 3 or more (new) H-bonds that mayalter structure.

3. It can change theconformational 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 phosphoryldonor links the energy status of the cell to the regulation of metabolism.

Kinases vary in specificity: dedicated and multifunctional kinases. Proteinkinase 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.

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 thenthe function of several proteins by Ser/Thr-phosphorylation.

cAMP activates PKA allosterically at 10 nM (activation mechanism is similarto 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 bindingrelieves 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 cellsor developmental stages.

Activation by specific proteolytic cleavage

Since ATP is not needed for this type of activation, Es outside the cell canalso 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 specificcleavage that makes the target pro-E active.

Examples:

- blood clotting cascade of proteolytic activations makes the response totrauma 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 ofcollagen is degraded in the uterus after delivery (procollagenase turns tocollagenase in a timely fashion)

-Programmed cell death, or apoptosis, is mediated by proteases calledcaspases generated from procaspases. Responding to certain signals (seeApoptosis lecture, next semester), caspases cause cell death throughoutmost of the animal kingdom (apoptosis gets rid of damaged or infectedcells 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 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

-the correct position of one of the N-Hs in the oxyanion hole is also takenonly after the above conformational changes occured

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 inthe zymogen while pretty structured in the mature E

- the oxyanion hole in the zymogen is too far from His 57 to promote thetetrahedral 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 hydrolyzesa Lys-Ile peptide bond in trypsinogen; small amount of trypsin is enough tospeed 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 theI 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 alreadyvery complementary to the AS

- the Lys 15-Ala 16 bond is eventually cleaved, but very slowly: the t1/2 ofE-I is several months

- the I is practically a S, too complementary to AS, binds too tightly andturns 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 (thatwould cause tissue damage and acute pancreatitis)

- there is also 1-antitrypsin (1-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 againsttissue damage weakens for smokers.