copyright (c) by w. h. freeman and company lecture no.4 enzymes: i] catalytic strategies (ch.9) ii]...

Copyright (c) by W. H. Freeman and Company

LECTURE No.4

Enzymes:

I] Catalytic Strategies (Ch.9)

II] Regulatory Strategies (Ch.10)


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


I] Catalytic strategies

Covalent catalysis and General acid-base catalysis: the example of

Chymotrypsin, a protease


Chymotrypsin cleaves peptides ”after” non-polar bulky residues


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


An unusually reactive Serine in Chymotrypsin, amongst 28


Chromogenic substrate analogues to measure activity


Kinetics of chymotrypsin


A covalent ES complex to explain the ”burst phase”


Active-site SER in binding-site pocket of Chymotrypsin


Why is Ser195 so reactive? The catalytic triad

Acid-base catalyst


Catalytic cycle of Chymotrypsin


Step 1: Substrate binding


Step 2: Nucleophilic attack on carbonyl carbon


Step 3: Acylation of Serine 195


Step 4 & 5: Peptide (amine) leaves, Water comes in


Step 6: Nucleophilic attack by water on the carbonyl carbon


Step 7: Peptide (carbonyl) leaves, Serine 195 regenerated


Stabilization of the tetrahedral intermediate


Hydrophobic pocket of Chymotrypsin: S1 pocket


More complex, more specific hydrophobic pockets of other proteases

Thrombin: Leu Val Pro Arg Gly Ser


Chymotrypsin (red) and Trypsin (blue): structurally similar enzymes


Structure of the S1 pockets explain substrate specificity


Subtilisin active site pocket

Subtilisin (Bacillus amyloliquefaciens)

Chymotrypsin


Structurally unrelated enzymes can develop identical strategies: convergent

evolution

Carboxypeptidase II from wheat


Site-directed mutagenesis to unravel the function of catalytic residues

Kcat reduced by a factor 106


Other proteases, other active sites...


Alternative residues for a common strategy: nucleophilic attack.


Structure of HIV protease II: an Aspartate protease


HIV protease inhibitor that mirrors the twofold symmetry of the enzyme


HIV protease – crixivan complex


Structural rearrangement upon binding of crixivan (Chain A)


I] Catalytic strategies

Metal ion catalysis: the example of Carbonic Anhydrase II, an enzyme with prodigious catalytic velocity


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...)


The active-site structure of human carbonic anhydrase II


Carbonic anhydrase activity is strongly pH-dependent

Active site group pKa close to 7.0


When bound to Zn(II), pKa of water drops from 15.7 to 7.0


Catalytic mechanism of carbonic anhydrase


A synthetic analog mimicks carbonic anhydrase catalytic mechanism

Water pKa=8.7

Hydration of CO2, 100-fold at pH 9.2


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 !


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!


Effect of buffer concentration on hydration of CO2


Histidine 64 shuttles protons from the active site to the buffer in solution


-carbonic anhydrases in archea: different structure but same function as carbonic

anhydrase II from humans


II] Regulatory Strategies

Allosteric control

(Isomerisation of enzymes:

”Isozymes”)

(Reversible covalent modifications)

(Proteolytic activation)


II] Regulatory strategies

Allosteric inhibition,”feedback” regulation:

the case of Aspartate Transcarbamoylase (ATCase)


Reaction catalyzed by ATCase

ATCase


Effect of cytidine triphosphate on ATCase activity (Gerhart & Pardee, 1962)


Modification of cysteine residues induces changes in ATCase structure


Changes in structure revealed by differencial sedimentation

(ultracentrifugation)

native p-HMB treated

Catalyticsubunit

Regulatorysubunit


Quaternary structure of ATCase(2C3 + 3R2) : ”Top-View”

(x 2)

Coordinated by 4x -SH


Quaternary structure of ATCase(2C3 + 3R2) : ”Side-View”


A bi-substrate analog to map the active-site residues


X-ray crystallography reveals the substrate-binding site

3x2 active sites / enzyme


Binding of PALA induces major conformational changes

(Tense, lower affinity) (Relaxed, higher affinity)


Molecular motion of the T-state to R-state transition


Binding sites of cytidine triphosphate (CTP,effector)

1x CTP binding-site per R unit

50Å away from catalytic site

How does CTP inhibits activity?


CTP induces a transition R T state by a concerted mechanism

[T]/[R]= 200


Allosteric enzymes do not follow Michaelis-Menten kinetics

Sigmoidal instead of hyperbolic


Two additive Michaelis-Menten kinetics: T state + R state.

Positive cooperativity!

Sum of the two hyperbolic curves


CTP an allosteric inhibitor of ATCase


ATP an allosteric activator of ATCase


Sequential models can account for allosteric effects

Several intermediate states can exist

Binding to one site influences affinity in neighboring site

Negative cooperativity


II] Regulatory Strategies

Hemoglobin: efficient O2 transport by positive cooperativity


Positive cooperativity enhances O2 delivery by hemoglobin

Hemoglobin increases by 1.7-fold the amount of oxygen delivered to the tissues


Oxygen binding site in hemoglobin is a prosthetic group: the heme


Non-planar porphyrin in deoxyhemoglobin


Conformational change of the heme upon O2 binding


Quaternary structure of hemoglobin: 2


T-state R-state transition in hemoglobin: structural rearrangement


O2 binding triggers a cascade of structural rearrangements


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


A natural allosteric inhibitor of hemoglobin: 2,3-BPG


2,3-BPG binds to the central cavity of deoxyhemoglobin (T state)

=> Reduces affinity for O2 in the T-state


Fetal hemoglobin presents a lower affinity for 2,3-BPG

2 -chains instead of 2 -chains

mutations HisSer in -chains

higher affinity for O2


Effect of pH and pCO2 on O2 release from hemoglobin: Bohr effect (1904)


Protons stabilize the quaternary structure of deoxyhemoglobin

Salt bridges at acidic pH, locks T-state conformation


Carbamylation of terminal amines by CO2

Negative charges at N-termini form new salt bridges

Stabilize deoxyhemoglobin: favors release of O2


Next Lecture (No.4)

Protein synthesis (Ch. 28)

Protein analyses (Ch. 4)


Remarks after the lecture

too long: 2h30 and section on hemoglobin not treated!

copyright (c) by w. h. freeman and company lecture no.4 enzymes: i] catalytic strategies (ch.9) ii]...

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