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Enzymatic Reactions

Ina L. UrbatschDepartment of Cell Biology and Biochemistry

TTUHSC

Office: 5C123E-mail: Ina.Urbatsch@ttuhsc.edu

Reading assignment for 10/27/2015:Meisenberg and Simmons, Chapter 4

Enzymatic ReactionsNutrients serve as starting material for metabolic processes. Enzymes catalyzethese metabolic processes. In our body thousands of metabolic intermediatesare formed and channeled though the different pathways in highly orderedfashion. Thousands of different enzymes are involved in different steps of thepathways. Without enzymes, life would not be possible.

General Outline: 1) Principles in Thermodynamics

2) Enzyme Kinetics

3) Regulation of Enzymatic Activity

(enzyme inhibition, covalent modifications)

4) Catalytic mechanisms

(chymotrypsin, ATP synthase)

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Thermodynamics(Greek: therme = heat + dynamics = movement)

Thermodynamics is the study of movement of energy from one system to another

The first law of thermodynamics: Energy is conservedEnergy is not lost and cannot be created but it can be interchanged between different forms e.g. heat (q), work (w), potential energy stored in chemical bonds, kinetic energy (random motion of molecules), etc.

Cells must be able to perform certain tasks in order to function properly. These tasks include:

- Synthesis of macromolecules- Transport of molecules across membranes- Cell movement.

Each of these tasks requires energy, which cells obtain via specific chemical reactions.

∆G <0 for a spontaneous reactionExergonic

∆G >0 if energy is needed to drive the reaction Endergonic

Gibbs Free Energy

∆G: Change in free energy, amount of free energy consumed or liberated during a chemical reaction.- Energy available to do work.- Predicts whether a reaction is favorable.

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A + B C + D

[product][substrate]

K’eq = =

Chemical reactions and equilibrium

The reaction equilibrium can be determined by letting the reaction proceed to completionand measuring the concentration of the reactants. The equilibrium constant defines the ratioof product to substrate concentrations at equilibrium.

[C] [D][A] [B]

under standard conditions: pH = 7, temperature at 25oC (298oK), solutes at 1M concentration, gases at 1 atm pressure.

Hydration of carbon dioxide (CO2)

Kequ. = = = [product]

[substrate][H2CO3][CO2]

1340

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∆G = ∆Go + RT ln [C] [D][A] [B]

∆Go = ∆Go’ under standard conditions: pH = 7, temperature at 25oC (298oK), solutes at 1M concentration, gases at 1 atm pressure.

At equilibrium ∆G = 0

∆Go’ = - RT ln [C] [D][A] [B]

The standard free energy change is related to the equilibrium constant:

∆Go’ = - RT ln K’equ

∆G <0 for a spontaneous reaction (exergonic)∆G =0 at equilibrium∆G >0 if energy is needed to drive the reaction

(endergonic)

Carbonic anhydrase in the red blood cells hydrates CO2 released from tissues,bicarbonate is pumped into the blood plasma by a specific membrane pump thatexchanges HCO3

- for Cl-. Removal of product HCO3- effectively leads to hydration

of more CO2.

In the lungs this process is reversed and CO2 exhaled.

∆G is dependent on product/substrate concentrations

∆G = ∆Go + RT ln [C] [D][A] [B]

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Thermodynamics and G tell us about the direction of a reaction but not about the rate of a reaction.

Kinetics: measures rates of chemical reactions.

Enzyme kinetics: measures rates of chemical reactions catalyzed by enzymes.

Example: k-2

k2

k-1

k1

E + S ES E + P

Kinetics

k: rate constants

Enzymes do not change the equilibrium of a chemical reaction but lower the energy of the transition state,e.g. they speed up or catalyze thermodynamically possible reactions.

Enzyme kinetics

Transition state

E + S E Sǂ E + P

Gǂ

Activation energy

ǂ

ESǂ

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Standard conditions:- pH is 7.0-temperature is 37oC

Rate enhancement by selected enzymes

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Enzymes are catalysts

The catalytic cycle. The substrate has to bind to the enzyme to form anoncovalent enzyme-substrate complex (enzyme • substrate). The actualreaction takes place while the substrate is bound to the enzyme.Note that the enzyme is regenerated at the end of the catalytic cycle.Therefore, only a small amount is needed to convert a large number ofsubstrates into product molecules.

Lock and key model

Induced-fit model

Most enzymes are globular proteins,substrate binds to the substrate binding pocket

called active site or catalytic site.

Enzymes are very selective for substrate and reaction type; in case ofa genetic defect only one reaction is blocked. Substrate specificity is determinedby the size, shape, and polar environment of the active site.

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Michaelis-Menten kinetics

V = Vmax

[S]

[S] + KM

1

V

1

Vmax

=Km

Vmax

+ x1

[S]

Lineweaver-Burk plot:Substrate concentration [S]

Rea

ctio

n V

eloc

ity (

V)

At low [S] velocity increases linear with [S].

At high [S] velocity is dependent on the [E],e.g. all enzymes are occupied with substrate.

Simplifications:- Only 1 substrate.- Initial rates when product formation is negligible.

E + S E Sǂ E + P

Michaelis-Menten kinetics

- Maximum velocity is reached when all the sites are filled.

- KM is the substrate concentration at Vmax/2, e.g. half of the active sites are filled.

- KM is a measure for the affinity of an enzyme for a particular substrate, e.g. high KM indicates weak binding, low KM indicates strong binding.

- Vmax reveals the turnover number also called kcat

- Kcat/KM is a measure for the catalytic efficiency.

Units: Vmax (μmol product formed/min/mg enzyme)Kcat (s-1), e.g. divide Vmax by mol/mg and express per secondKM usually (mM) or (μM)Kcat/KM (s-1 M-1)

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KM is a measure for the affinity of the active site

One of the fastest enzymes known

Maximal turnover numbers of some enzymes

Under physiological conditions mostenzymes are not saturated withsubstrates, therefore kcat/KM is betterpredictor of actual rates.

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Kinetic perfection:if kcat/KM is close to the diffusion limit ~ 108-109 (s-1M-1)

Kinetics of substrate decay

Zero order kinetics

First order kinetics

First order kinetics: one substrate, reactionrate is directly proportional to the substrateconcentration which declines over time becauseless and less substrate is available.

Second order kinetics: two substrates,reaction rate depends on concentration of bothsubstrates.

Zero order kinetics: independent of theconcentration of substrate.

A. Drug metabolism (liver) usually follows first

order kinetics ([S] < Km). T1/2 = half of S is

consumed.

B. Metabolism of alcohol is limited by alcoholdehydrogenase which becomes rapidlyoversaturated after initial ingestion (zero orderkinetics) .

A

B

B) Blood alcohol concentration after the ingestion of 120 g of ethanol. The linear decrease of the alcohol level 2 to 10 hours after ingestion shows that a zero-order reaction limits the rate of alcohol metabolism. GI, gastrointestinal.

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Regulation of enzymatic activity

• To maintain homeostasis, the rates of various reactions must be regulated. This is accomplished by regulating the enzymes that catalyze the reactions.

• Some of the mechanisms of regulation include:

- Reversible inhibition

- Irreversible inhibition

- Covalent modification

- Allosteric regulation (e.g. Hemoglobin)

- Regulation of enzyme expression (transcription and translation)

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Competitive inhibitors structurally resemble

the substrate and bind to the active site

bind to allosteric sites

only bind to enzyme-substrate

complex

Reversible enzyme inhibition

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Competitive inhibitors

Lineweaver Burk blot

Presence of inhibitor increases Km but Vmax is unchanged

Role of alcohol dehydrogenase (ADH) in the metabolism of methanol and ethanol. The two substrates compete for the enzyme. Therefore, ethanol inhibits the formation

of toxic formaldehyde and formic acid from methanol.

Competitive inhibitors

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Dihydrofolate reductase plays a role in the synthesis of purines and pyrimidines

Competitive inhibitors

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HIV protease in complex with the inhibitor Crixivan

Crixivan mimics a peptidesubstrate but binds verytightly to the active site (slow“off-rate”).It specifically interacts with 2Asp at the bottom of the site.

flaps

Asp Asp

Noncompetitive inhibitors

Lineweaver Burk blot

Presence of inhibitor decreases Vmax but Km is unchanged

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O CH3

F P-O-CH

CH3 CH3

O CH3

P-O-CH

CH3 CH3

Sarin(nerve gas)

Irreversible enzyme inhibitionby covalent modification

More potent to insects than mammals:

Potent nerve poison:

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Bacterial cell wall with sugars in yellow, tetrapeptides in red, pentaglycine in blue

Penicillin irreversibly inhibits glycopeptide transpeptidase which adds the first peptide (red triangle) to the pentaglycine residues (blues circles)

Penicillin

Penicillin

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Regulation by covalent attachments

Farnesylation: lipid anchor to attach Ras to the plasma membrane surface

Many enzymes are regulated by phosphorylationPhosphorylation is very effective means of cell cycle control

Ubiquitination signals that a protein is to be destroyed

-phosphate is transferred from ATP to Ser, Thr, or Tyr residues within a specific recognition sequence,

e.g. consensus sequence in PKA is RRxSZ or RRxTZx is small, Z is large hydrophobic

Usually:

Enzyme phosphorylation by kinases:

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Dephosphorylation:

Note: Phosphorylation - dephosphorylation is not a reverse process, two different enzymes are required and

in both reactions free energy is generated

∆G = -12 kcal/mol

Highly favorable free energy changes ensure unidirectional P -transfer

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Phosphorylation controls the activity of the target proteins:

- adds two negative charges

- Phosphate group can form three or more hydrogen bonds

- half of the ∆G (-12 kcal/mol) goes into the P -bond, other half is conserved in the protein

- effects can be highly amplified: a single activated kinase can phosphorylate hundreds of targets

- ATP links the energy status of the cell to the phosphorylation

Class Type of reaction Example

1. Oxidoreductases Oxidation-reduction Alcohol dehydrolgenase

2. Transferases Group transferProtein kinaseATP synthase

3. HydrolasesHydrolysis reaction (transfer of groups to water)

Chymotrypsin

4. LyasesAddition or removal of groups to form double bonds

Fumerase

5. IsomerasesIsomerization (intramolecular group transfer)

Triphosphate isomerase

6. LigasesLigation of two substrates at the expense of ATP hydrolysis

Aminoacyl-tRNAsynthase

Six major classes of enzymes

The suffix “-ase” always indicates an enzyme; other common suffix is “-in” (e.g. trypsin, fibrin)

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ATP synthaseMyosin (molecular motor)Adenylate kinaseTransducin (signal transduction)recA (DNA repair)Elongation factor Tu

ABC transporter

Transducin is the G-protein that interacts with rhodopsin during light reception, and is a GTPase not an ATPase. Thus, specificity can be ‘tuned’ to discriminate between ATP and GTP.

Core domain is found in many Kinases and ATP hydrolyzing enzymes:

P-loop

P-loop: GxxGxGKS/Talso called Walker A motif

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Mg2+

OH2

H2O

H2O

OH2

Mg - neutralizes some charges on --phosphate- holds nucleotide in well defined conformation- increases binding energy- stabilizes the transition state

Essential cofactor Mg2+

cytoplasm

periplasm

ATP Synthase

F1

Fo

ATP

ADP + Pi

H+

H+

a

b2

c10-14

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Nucleotide Binding Sites on F1-ATPase

3 catalytic sites

mainly on subunits, on interface to

3 noncatalytic sites

mainly on subunits, on interface to

Transition state in F1FO-ATP synthase

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ATP Hydrolysis Causes Subunit Rotation

fluorescent actin filament

biotin streptavidin

biotin

F1 (33, F1Fo)

modified from: Kinosita et al. (2000) Phil. Trans. Soc. Lond. B 355, 473-489

His-tags

Ni2+ support

ATP

ADP + Pi

-Highly selective catalysts, usually are specific for one or a few substrates.

-Do not change the concentrations of substrates and products at equilibrium (i.e. Keq) but they decrease the time required to reach equilibrium.

-Catalyze reactions by stabilizing the transition state,but do not change the thermodynamics of chemical reactions.

-Bring substrates together in optimal orientation in the active site.

Enzymes