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Transcript of Copyright (c) by W. H. Freeman and Company LECTURE No.4 Enzymes: I] Catalytic Strategies (Ch.9) II]...
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Copyright (c) by W. H. Freeman and Company
LECTURE No.4
Enzymes:
I] Catalytic Strategies (Ch.9)
II] Regulatory Strategies (Ch.10)
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A few basic catalytic principles used by many enzymes
Covalent catalysis: transient covalent bond between enzyme and substrate
General acid-base catalysis: other molecule than water gives/accept protons (Histidine)
Metal ion catalysis: several strategies possible
Catalysis by approximation: bringing substrates in proximity
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I] Catalytic strategies
Covalent catalysis and General acid-base catalysis: the example of
Chymotrypsin, a protease
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Chymotrypsin cleaves peptides ”after” non-polar bulky residues
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Chymotrypsin facilitates nucleophylic attack
Amide bond hydrolysis is thermodynamically favored but very slow
Carbon in carbonyl group resistant to nucleophilic attack: partial double-bond with N & planar geometry
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An unusually reactive Serine in Chymotrypsin, amongst 28
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Chromogenic substrate analogues to measure activity
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Kinetics of chymotrypsin
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A covalent ES complex to explain the ”burst phase”
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Active-site SER in binding-site pocket of Chymotrypsin
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Why is Ser195 so reactive? The catalytic triad
Acid-base catalyst
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Catalytic cycle of Chymotrypsin
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Step 1: Substrate binding
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Step 2: Nucleophilic attack on carbonyl carbon
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Step 3: Acylation of Serine 195
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Step 4 & 5: Peptide (amine) leaves, Water comes in
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Step 6: Nucleophilic attack by water on the carbonyl carbon
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Step 7: Peptide (carbonyl) leaves, Serine 195 regenerated
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Stabilization of the tetrahedral intermediate
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Hydrophobic pocket of Chymotrypsin: S1 pocket
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More complex, more specific hydrophobic pockets of other proteases
Thrombin: Leu Val Pro Arg Gly Ser
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Chymotrypsin (red) and Trypsin (blue): structurally similar enzymes
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Structure of the S1 pockets explain substrate specificity
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Subtilisin active site pocket
Subtilisin (Bacillus amyloliquefaciens)
Chymotrypsin
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Structurally unrelated enzymes can develop identical strategies: convergent
evolution
Carboxypeptidase II from wheat
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Site-directed mutagenesis to unravel the function of catalytic residues
Kcat reduced by a factor 106
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Other proteases, other active sites...
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Alternative residues for a common strategy: nucleophilic attack.
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Structure of HIV protease II: an Aspartate protease
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HIV protease inhibitor that mirrors the twofold symmetry of the enzyme
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HIV protease – crixivan complex
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Structural rearrangement upon binding of crixivan (Chain A)
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I] Catalytic strategies
Metal ion catalysis: the example of Carbonic Anhydrase II, an enzyme with prodigious catalytic velocity
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Hydration of CO2 in the blood
Non catalyzed reaction happens at moderate pace: k1=0.15 s-1 (pH7.0, 37°C)
Carbonic anhydrase: Kcat=600.000 s-1
Special strategies to compensate for limiting factors (diffusion limits...)
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The active-site structure of human carbonic anhydrase II
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Carbonic anhydrase activity is strongly pH-dependent
Active site group pKa close to 7.0
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When bound to Zn(II), pKa of water drops from 15.7 to 7.0
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Catalytic mechanism of carbonic anhydrase
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A synthetic analog mimicks carbonic anhydrase catalytic mechanism
Water pKa=8.7
Hydration of CO2, 100-fold at pH 9.2
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Kinetics of water deprotonation illustrates rate constants limitation
Proton diffusion: k= 10-11 M-1 s-1
In above reaction k-1 ≤ 1011 M-1 s-1
at pH7.0, K=10-7 M => k1 ≤ 104 s-1
Problem: kcat= 106 s-1 !
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Buffers displace the equilibrium constant
Rate of proton loss is given by [B].k1´
Buffer diffusion: k= 109 M-1 s-1
With [B]=10-3 M, [B]. k1´= 10-3 x109=10-6 s-1
Problem: buffers not accessible to active site!
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Effect of buffer concentration on hydration of CO2
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Histidine 64 shuttles protons from the active site to the buffer in solution
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-carbonic anhydrases in archea: different structure but same function as carbonic
anhydrase II from humans
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II] Regulatory Strategies
Allosteric control
(Isomerisation of enzymes:
”Isozymes”)
(Reversible covalent modifications)
(Proteolytic activation)
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II] Regulatory strategies
Allosteric inhibition,”feedback” regulation:
the case of Aspartate Transcarbamoylase (ATCase)
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Reaction catalyzed by ATCase
ATCase
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Effect of cytidine triphosphate on ATCase activity (Gerhart & Pardee, 1962)
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Modification of cysteine residues induces changes in ATCase structure
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Changes in structure revealed by differencial sedimentation
(ultracentrifugation)
native p-HMB treated
Catalyticsubunit
Regulatorysubunit
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Quaternary structure of ATCase(2C3 + 3R2) : ”Top-View”
(x 2)
Coordinated by 4x -SH
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Quaternary structure of ATCase(2C3 + 3R2) : ”Side-View”
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A bi-substrate analog to map the active-site residues
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X-ray crystallography reveals the substrate-binding site
3x2 active sites / enzyme
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Binding of PALA induces major conformational changes
(Tense, lower affinity) (Relaxed, higher affinity)
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Molecular motion of the T-state to R-state transition
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Binding sites of cytidine triphosphate (CTP,effector)
1x CTP binding-site per R unit
50Å away from catalytic site
How does CTP inhibits activity?
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CTP induces a transition R T state by a concerted mechanism
[T]/[R]= 200
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Allosteric enzymes do not follow Michaelis-Menten kinetics
Sigmoidal instead of hyperbolic
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Two additive Michaelis-Menten kinetics: T state + R state.
Positive cooperativity!
Sum of the two hyperbolic curves
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CTP an allosteric inhibitor of ATCase
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ATP an allosteric activator of ATCase
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Sequential models can account for allosteric effects
Several intermediate states can exist
Binding to one site influences affinity in neighboring site
Negative cooperativity
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II] Regulatory Strategies
Hemoglobin: efficient O2 transport by positive cooperativity
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Positive cooperativity enhances O2 delivery by hemoglobin
Hemoglobin increases by 1.7-fold the amount of oxygen delivered to the tissues
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Oxygen binding site in hemoglobin is a prosthetic group: the heme
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Non-planar porphyrin in deoxyhemoglobin
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Conformational change of the heme upon O2 binding
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Quaternary structure of hemoglobin: 2
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T-state R-state transition in hemoglobin: structural rearrangement
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O2 binding triggers a cascade of structural rearrangements
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Concerted or Sequential cooperativity for hemoglobin?
Both!
3 sites occupied: R-state with 4th site having 20-fold higher affinity for O2
1 site occupied: T-state with other sites having 4-fold higher affinity for O2
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A natural allosteric inhibitor of hemoglobin: 2,3-BPG
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2,3-BPG binds to the central cavity of deoxyhemoglobin (T state)
=> Reduces affinity for O2 in the T-state
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Fetal hemoglobin presents a lower affinity for 2,3-BPG
2 -chains instead of 2 -chains
mutations HisSer in -chains
higher affinity for O2
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Effect of pH and pCO2 on O2 release from hemoglobin: Bohr effect (1904)
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Protons stabilize the quaternary structure of deoxyhemoglobin
Salt bridges at acidic pH, locks T-state conformation
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Carbamylation of terminal amines by CO2
Negative charges at N-termini form new salt bridges
Stabilize deoxyhemoglobin: favors release of O2
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Next Lecture (No.4)
Protein synthesis (Ch. 28)
Protein analyses (Ch. 4)
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Remarks after the lecture
too long: 2h30 and section on hemoglobin not treated!