Regulatory strategies Attila Ambrus

1. Regulatory strategies Attila Ambrus

2. aspartate trans- carbamoylase first step in pyrimidine biosynthesis

3. 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)

4. 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).

5. 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).

6. 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

7. 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)

8. 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:

9. 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.

10. 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

11. 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).

12. 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!

13. concerted mechanism

16. 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]

17. 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).

18. 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.

19. 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.

20. 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.

21. 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.

23. 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:

24. 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).

25. 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

26. 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)

27. 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.