Enzyme regulation 1. Modulation of catalytic properties a. Substrate concentration b. cooperativity (allostery) 2. Post-traductionnal modifications c1. irreversibles (proteolysis) c2. reversibles (phosphorylation) 3. Isoenzymes (separate genes, alternative splicing) 4. Multi-functionnal enzymes, multi-enzymatic complexes « channeling » 5. Modification of enzyme concentration Synthesis, degradation
S08d REPLIEMENTE DES PROTEINES DANS LA CELLULE
This illustration shows a cross-section through the blood, with blood serum in the upper half and a red blood cell in the lower half. In the serum, look for Y-shaped antibodies, long thin fibrinogen molecules (in light red) and many small albumin proteins. The large UFO-shaped objects are low density lipoprotein and the six-armed protein is complement C1. The red blood cell is filled with hemoglobin, in red. The cell wall, in purple, is braced on the inner surface by long spectrin chains connected at one end to a small segment of actin filament. Which in cellular terms in this macrophage looks like:
S08d REPLIEMENTE DES PROTEINES DANS LA CELLULE
Plus compliqué dans la cellule à cause de l’encombrement (crowding en anglais) protéines 250-300 mg/ml This illustration shows a cross-section of a small portion of an Escherichia coli cell. The cell wall, with two concentric membranes studded with transmembrane proteins, is shown in green. A large flagellar motor crosses the entire wall, turning the flagellum that extends upwards from the surface. The cytoplasmic area is colored blue and purple. The large purple molecules are ribosomes and the small, L-shaped maroon molecules are tRNA, and the white strands are mRNA. Enzymes are shown in blue. The nucleoid region is shown in yellow and orange, with the long DNA circle shown in yellow, wrapped around HU protein (bacterial nucleosomes). In the center of the nucleoid region shown here, you might find a replication fork, with DNA polymerase (in red-orange) replicating new DNA. © David S. Goodsell 1999.
Macromolecular crowding: obvious but underappreciated. Ellis RJ. Trends Biochem Sci. 2001 26, 597604. Review.
S08d REPLIEMENTE DES PROTEINES DANS LA CELLULE
L’encombrement augmente l’affinité par le jeu des « activités » Des activités qui ne peuvent plus être aproximées par les concentrations
Fig. 3. The highly nonlinear characteristics of macromolecular crowding. (a) The dependency of activity coefficient on concentration of crowding agent. (b) Estimated activity coefficients for spherical molecules of increasing molecular weight in the cytoplasm of Escherichia coli. The curves are drawn for two specific volumes. (c) Dependence of reaction rate constant on the degree of crowding in cases where the reaction is either diffusionlimited (green curve) or transition state-limited (blue curve). The reaction rate is transition-state limited at low degrees of crowding and diffusion-limited at high degrees of crowding (red curve). Note that the reaction rate constant in (c) is represented on a log scale. Reproduced, with permission, from [15, 3 and 13], respectively.
Fig. 2. The importance of size in volume exclusion. The squares define volumes containing spherical macromolecules occupying 30% of the available space. (a) The centre of an introduced small molecule has access to virtually all of the remaining 70% of the space, indicated in yellow. (b) The centre of an introduced molecule similar in size to the macromolecules is excluded from most of this 70% as it cannot approach these macromolecules to a distance less than that indicated by the open circles.
Macroviscogens: proteins, PEG Decrease the accessible volume, do not decrease the diffusion rate Microviscogens: sucrose, glycerol decrease the diffusion rate
s03. Structure primaire des protéines et modifications posttraductionnelles F. Crick 1959
DNA
DNA
5’ process ribosome
mRNA 3’ mature mRNA
mRNA cap 5’
3’ tail
proteins proteins
Prokaryote Student dormitory
Eukaryote Furnished apartment
Structure primaire des protéines et modifications post-traductionnelles
1948: 1 gène correspond à 1 enzyme
Repliement (obligatoire) Modification éventuelles Transport éventuel
Protéine native
1 séquence correspond à 1 structure native (avec quelques exceptions)
Régulation de l’activité enzymatique
Figure 2-88. Glycolysis and the citric acid cycle are at the center of metabolism. Some 500 metabolic reactions of a typical cell are shown schematically with the reactions of glycolysis and the citric acid cycle in red. Other reactions either lead into these two central pathways—delivering small molecules to be catabolized with production of energy—or they lead outward and thereby supply carbon compounds for the purpose of biosynthesis.
Régulation de l’activité enzymatique
Hierarchical regulation Adaptation
Time
Substrate concentration
Intracellular
« Instantaneous »
Allosteric regulation
Intracellular, Metabolism integration
very rapid ms to s
Covalent modification
Extracellular (hormons)
Rapid s to min
Expression gene control
intra- and extracellular
Slow (min to days)
Régulation de l’activité enzymatique: 1A. Concentration du substrat L’équation de Michaelis n’est pas applicable en général pour la cellule sans une analyse critique 1. [ E ] n’est pas >> [ s ] 2. [ P ] n’est pas négligeable
Régulation de l’activité enzymatique: 1A. Concentration du substrat
Régulation de l’activité enzymatique: 1A. Concentration du substrat
PFK F1,6BPase Aldolase TPI GAPDH PGK PK LDH
active site substrate Products AS/Sfr µM % free total,µM free tot free 6.4 25 F6P 1500 1495 0.004 2.1 14 F1,6BP 80 8.9 0.23 216 50 F1,6BP 80 8.9 DHAP 160 116 24 GA3P 80 17.8 17.8 0.5 60 77 DHAP 160 116 GA3P 80 373 73 GA3P 80 17.8 1,3-BPG 50 0.14 21 35 82 1,3-BPG 50 0.14 3-PGA 200 196 247 46 59 PEP 65 46.9 Pyruvate 380 360 1.0 79 52 Pyruvate 380 360 Lactate 3700 3655 0.22
Régulation de l’activité enzymatique: 1A. Concentration du substrat Concentration cellulaire des substrats sont en général proches des Km: enzymes de la glycolyse Enzyme
Source
Substrat
Conc (µM) Km (µM)
Km/[s]
Glucose-6-P Isomérase
Muscle
G-6-P F-6-P
450 110
700 120
1.6 1.1
Aldolase
Muscle
FBP
32
100
3.1
Triosephosphate Isomérase
Muscle
G-3-P DHAP
3 50
460 870
153 17
GAPDH
Muscle
G-3-P NAD
3 600
70 70
23 0.08
PGK
Muscle
3-PG ADP
60 600
1200 350
200 0.6
PGM
Muscle
3-PG
60
5000
83
Enolase
Muscle
2-PG
7
70
10
Différents enzymes
ATP, NAD(H)
0.001 - 1
Régulation de l’activité enzymatique
Régulation de la Glycolyse Regulation des 3 étapes irréversibles et LIMITANTES DE VITESSE
Régulation de l’activité enzymatique Si la PFK-1 et la F-1,6-BPase-1 fonctionne en même temps: Hydrolyse de l’ATP Exemple de CYCLE FUTILE (inutile, mais….. Produis de la chaleur!)
F-2,6-BP Phosphorylation
Régulation de l’activité enzymatique Futile cycles may have 2 roles:
1) Amplification of signals Regulation is more efficient than starting from zero
2) Generation of heat ATP + H2O
ADP + Pi + H+ + heat
Estimation of the Fructose Diphosphatase-Phosphofructokinase Substrate Cycle in the Flight Muscle of Bombus affinis By MICHAEL G. CLARK, DAVID P. BLOXHAM, PAUL C. HOLLAND and HENRY A. LARDY Institute for Enzyme Research and the Department of Biochemistry, University of Wisconsin, 1710 University Avenue, Madison, Wis. 53706, U.S.A. (Received 29 December 1972)
Bombus cannot fly if its temperature is less than 20°C. It is heated by PFK/F1,6BPase futile cycle Substrate cycling of fructose 6-phosphate through reactions catalysed by phosphofructokinase and fructose diphosphatase was estimated in bumble-bee (Bombus affinis) flight muscle in vivo. Estimations of substrate cycling of fructose 6-phosphate and of glycolysis were made from the equilibrium value of the 3H/14C ratio in glucose 6-phosphate as well as the rate of 3H release to water after the metabolism of [5-3H,U-14C]glucose. In flight, the metabolism of glucose proceeded exclusively through glycolysis (20.4 µmol/min per g fresh wt.) and there was no evidence for substrate cycling. In the resting bumble-bee exposed to low temperatures (5°C), the pattern of glucose metabolism in the flight muscle was altered so that substrate cycling was high (10.4/miol/min per g fresh wt.) and glycolysis was decreased (5.8 µmol/min per g fresh wt.). 5. The rate of substrate cycling in the resting bumble-bee flight muscle was inversely related to the ambient temperature, since at 27°, 21° and 5°C the rates of substrate cycling were 0, 0.48 and 10.4 µmol/min per g fresh wt. respectively.
Malignant hyperthermia (MH or MHS for "malignant hyperthermia syndrome", or "malignant hyperpyrexia due to anaesthesia") is a rare life-threatening condition that is triggered by exposure to certain drugs used for general anesthesia The disease appears in pigs under stress
Accelerated substrate cycling of fructose-6-phosphate in the muscle of malignant hyperthermic pigs. Clark MG, Williams CH, Pfeifer WF, Bloxham DP, Holland PC, Taylor CA, Lardy HA. Nature. 1973, 245(5420):99-101.
Régulation de l’activité enzymatique: Modifications post-traductionnelles
Régulation de la Pyruvate kinase HEPATIQUE par le rapport Insuline-glucagon; la PK musculaire n’est pas régulé (isoenzyme)
1. Modulation of catalytic properties
Régulation de l’activité enzymatique: 1A. Concentration du substrat Vmax [ S] vi =
Km + [ S]
Résultat: concentrations STATIONNAIRES des métabolites
Increase of v with [s] Km = 100 µM; Vmax = 5 µmol/min/ml [ s ] = 10 µM [ s ] = 20 µM [ s ] = 100 µM [ s ] = 200 µM
v = 0.45 µmol/min/ml v = 0.83 µmol/min/ml v = 2.5 µmol/min/ml v = 3.33 µmol/min/ml
[ s ] = 500 µM v = 4.16 µmol/min/ml [ s ] = 1000 µM v = 4.54 µmol/min/ml
1.84 x
1.33 x
1.09 x
2. Post-traductionnal modifications
Régulation de l’activité enzymatique F. Crick 1959
Régulation de l’activité enzymatique
1948: 1 gene encodes 1 enzyme
Folding (OBLIGATOIRY) MODIFICATION TRANSPORT
Enzyme (native) DEGRADATION
Amino acides, peptides
1 sequence « encodes » 1 native structure
Régulation de l’activité enzymatique: Modifications post-traductionnelles Exemple de la glutamine synthétase
Régulation de l’activité enzymatique: Modifications post-traductionnelles
Régulation de l’activité enzymatique: Modifications post-traductionnelles
La Glutamine synthétase est Un dodécamère
Régulation de l’activité enzymatique: Modifications post-traductionnelles
Examples of cellular processes regulated by Ser/Thr protein kinases and the signals to which they respond.
Phosphorylation and dephosphorylation are not the reverse of each other: the net result is conversion of ATP into ADP + Pi (∆G = -12 kcal/mol). The rate of cycling depends on the relative activities of the kinases and phosphatases involved.
Phosphorylation of proteins: • changes electrostatic interactions by addition of two negative charges per phosphate; • may result in 3 H-bonds per phosphate; • changes the free energy of a protein with appr. -6 kcal/mol; this energy can be used for conformational changes; • is kinetically controlled (can be slow or fast, depending on the activity and amount of kinases and phosphatases); • can evoke amplified effects: a single kinase can phosphorylate hundreds of target proteins (e.g. protein kinase A); • is linked to the energy status of a cell (as ATP is the phosphate donor).
• Protein kinases are a very large family of enzymes (559 homologues in human): • Dedicated protein kinases: phosphorylate single proteins or groups of related proteins • Multifunctional protein kinases: have very different target molecules
• Many protein kinases recognize the target motif: Arg - Arg - X - (Ser/Thr) - Z (Ser or Thr being the site of phosphorylation, X a small residue and Z a large hydrophobic residue)
Example: protein kinase A Protein kinase A modulates the activity of many proteins by phosphorylation. Protein kinase A occurs in an inactive form (R2C2), consisting of two regulatory (inhibiting) subunits and two catalytic subunits, and in an active form (2C). cAMP activates protein kinase A by dissociation of the R-subunits from the R2C2complex (allosteric activation).
The R-subunits contain a pseudosubstrate sequence: Arg - Arg - Gly - Ala - Ile, which binds to the catalytic site of the C-subunits. Binding of cAMP allosterically moves the pseudosubstrate sequence out of the catalytic sites.
Protein kinases bind ATP and target protein in a cleft. ATP and the inhibitor bind in a deep cleft between two lobes of protein kinase A: • one lobe binds ATP-Mg2+; • the other lobe binds the inhibitor and contains the catalytic residues; • the arginines of the inhibitor bind to carboxylates in PKA; the nonpolar Z-residue binds in a hydrophobic groove in PKA; • large part of this structure is conserved in other protein kinases.
3. Isoenzymes
Régulation de l’activité enzymatique: 2. ISOENZYMES
Isoenzymes = catalyze the same reaction but proteins are different Very often encoded separate genes; Sometimes alternative splicing Different tissue localization (Lactate DH) Different cellular localization (Malate DH) Sometimes different regulation (muscle and liver Pyruvate kinase) Very often the significance is not clear (similar kinetic properties)
2. ISOENZYMES: LDH
2. ISOENZYMES: LDH
Figure 10.27. Isozymes of Lactate Dehydrogenase. (A) The rat heart LDH isozyme profile changes in the course of development. The H isozyme is represented by squares and the M isozyme by circles. The negative and positive numbers denote the days before and after birth, respectively. (B) LDH isozyme content varies by tissue. [(A) After W.-H. Li, Molecular Evolution (Sinauer, 1997), p. 283; (B) After K. Urich, Comparative Animal Biochemistry (Springer Verlag, 1990), p. 542.]
2. ISOENZYMES: LDH
http://www.web.virginia.edu/Heidi/home.htm
2. ISOENZYMES: LDH
Hybrid formation by isoenzymes: subunits should be identical and interfaces should be identical
Rms 0.053 nm subunit
1 nm = 10 A
Rms 0.074 nm tetramer
2. ISOENZYMES: MDH
2. ISOENZYMES: MDH
Comparison of human Malate DH Mitochondrial and Cytosolic Little sequence similarity Structure are identical RULE: structure is more conserved than sequence (during evolution selection is done by function)
Régulation de l’activité enzymatique: 2. Isoenzymes
HK:
non spécifique pour le glucose constitutive 1cza HK Km approx 100 µM
3FGU GK
GK
Spécifique pour le glucose Inductible (contrôle hormonal) Km approx 5 mM
4. Multi-functionnal enzymes
Régulation de l’activité enzymatique: Enzymes bi-fonctionnelles
PFK-2/F-2,6-BPase-2
Domain Structure of the Bifunctional Enzyme Phosphofructokinase 2. The kinase domain (purple) is fused to the phosphatase domain (red). The kinase domain is a P-loop NTP hydrolase domain, as indicated by the purple shading (Section 9.4.4). The bar represents the amino acid sequence of the enzyme.
Régulation de l’activité enzymatique: Enzymes bi-fonctionnelles
Sous-unité α
Sous-unité β
Régulation de l’activité enzymatique: Complexes multi-enzymatiques
Figure 3-54. The structure of pyruvate dehydrogenase. This enzyme complex catalyzes the conversion of pyruvate to acetyl CoA, as part of the pathway that oxidizes sugars to CO2 and H2O. It is an example of a large multienzyme complex in which reaction intermediates are passed directly from one enzyme to another.
Régulation de l’activité enzymatique: Complexes multi-enzymatiques
Régulation de l’activité enzymatique: Complexes multi-enzymatiques
Le Complexe PDH
Permèt le passage direct d’une enzyme à la suivante sans dissociation CANALISATION (channeling)
5. Modification of enzyme concentration Synthesis, degradation
Régulation de l’activité enzymatique: Contrôle par la vitesse de synthèse
Régulation de l’activité enzymatique: Contrôle par la vitesse de synthèse 3) Glucagon and insulin mediate long-term effects by inducing and repressing the synthesis of key enzymes. Glucagon induces the synthesis of: PEP-carboxykinase Gluconeogenic
fructose 1,6-bisphosphatase
enzymes
glucose 6-phosphatase certain aminotransferases
Glucagon represses the synthesis of:
glucokinase Glycolytic
PFK1
enzymes
pyruvate kinase
Insulin generally opposes these actions.
Régulation de l’activité enzymatique: Contrôle par la vitesse de dégradation
Table 23.2. Processes regulated by protein degradation *Gene transcription *Cell-cycle progression Organ formation Circadian rhythms Inflammatory response *Tumor suppression Cholesterol metabolism *Antigen processing
Régulation de l’activité enzymatique: Contrôle par la vitesse de dégradation
Règle N-terminale
AA stabilisants
AA destabilisants (t1/2 = 2 to 30 minutes) Arg His Ile Leu Lys Phe Trp Tyr
(t1/2 > 20 hours) Ala Cys Gly Met Pro Ser Thr Val
Destabilisation après modification chimique (t1/2 = 3 to 30 minutes) Asn Gln
Asp Glu
FIN
Régulation de l’activité enzymatique
1. Modulation des propriétés catalytiques a. Concentration de substrat b. Coopérativité (allostérie) c. Modifications post-traductionnelles c1. irreversibles (protéolyse) c2. reversibles (phosphorylation, etc.) 2. Isoenzymes 3. « Canalisation »: Enzymes multi-fonctionnelles, complexes multi-enzymatiques 4. Modification de la concentration en enzyme Synthèse, dégradation
