BCOR 011 Lecture 12 9/28/2005

ENZYMESENZYMES

Last time…

-G reaction “can” go spontaneous

But when will it go?

And at what rate?

Thermodynamics

Whether a reaction will occur

Kinetics

WHEN a reaction will occur

What governs WHEN a reaction will occur?

The tower of blocks falling is favorablebut when will it happen?

Oxidation of carbohydrate polymers (starch)to carbon dioxide and water is favorablebut when will it happen?

Gasoline burning to carbon dioxide and wateris favorablebut when will it happen ?

For a Reaction to occur need to For a Reaction to occur need to Destabilize Existing StateDestabilize Existing Stateto to INPUT ENERGYINPUT ENERGY

Now In Transition

Potentialnet

usableenergy

Destabilizationenergy input“Activation

Energy”

Potentialnet

usableenergy

Need to INPUT ENERGY to Destabilize Existing State

In Transition After

Potentialnet

usableenergy

netusableenergy

released

RegainActivation

EnergyInvested

What does activation energy represent?

For a Reaction to Occur…

- reactants must find each other, - meet in proper orientation - and hit with sufficient force

ProductiveCollision

ManyNon-productive

Collisions

Needs of Typical chemical reactions

- need large number of molecular collisions

- need collide violently enough to break pre-existing bonds (not bounce)

- need high concentration to find eachother at significant rate

HEAT !

The energy profile for an exergonic reaction

Fre

e en

ergy

Progress of the reaction

∆G < O

EA

Figure 8.14

A B

C D

Reactants

A

C D

B

Transition state

A B

C D

Products

Molecules withsufficient

Energy (<5%)

Molecules withsufficient

Energy (~40%)

Temp 1 Temp 2 EA

ENZYMES make reactionseasier to occur at

reasonable temperatureby

LOWERING LOWERING the the ACTIVATION ENERGYACTIVATION ENERGY

EEAAof the reactionof the reaction

Activation EnergyEnergy necessary to overcome the status quo

G Thermodynamic“favorablility”

EA

“ease” of initiating reaction

G

EA

CATALYSTS:

promote a specific reactionBut are NOT consumed in the process

Key concepts:

Promotes - does not alter what would normally occur thermodynamically

Specificity - promotes only one reaction, only between specific reactants to give specific products

Reusable - regenerated in the process

ENZYMES are biological CATALYSTS

- usually PROTEINS

- sometimes RNA or RNA/protein complexes

Hard path

Easy path

Enzymes work as catalysts by providing an easy path to the same point

HOW?

How do Enzymes do it?

1. Enzymes have BINDING AFFINITYfor their reactants = Substrates

Brings substrates in close proximity: conc

Enzymes act as a Specific Platform

Have a very Specific 3-D Shape

With a Specific Arrangementof Functional Groups

FlexibleOH

HO

+

HO

PolarPolar

NonpolarNonpolar

ChargedCharged

Stabilized InteractionsStabilized Interactions

-

HO

OH

HO

OH

+

HO

OH

SPECIFICITYSPECIFICITY is the Key to Enzyme Action is the Key to Enzyme Action

ENZYMES:

Bind ONLY specific things

Bind them ONLY in a Specific 3-D Orientation

2. Enzymes ORIENT Substrates always in productive orientation

ProductiveCollision

ManyNon-productive

Collisions

ONLY ProductiveCollisions

-

HO

OH

HO

OH

+

HO

OH

With just a little nudge, can’t help but react

3a. Physical Strain3a. Physical Strain

3b.Chemical Strain3b.Chemical Strain

3. Enzymes cause BOND STRAINBOND STRAIN - destabilize existing bonds- destabilize existing bonds

““nutcracker effect”nutcracker effect”

The active site– Is the region on the enzyme where

the substrate binds

Figure 8.16

Substate

Active site

Enzyme

(a)

Induced fit of a substrate

Figure 8.16 (b)

Enzyme- substratecomplex

Enzyme-substrate interactions

Fischer: Lock & key

Koshland:Induced fit

3a. Physical bond strain3a. Physical bond strainDraw an quarter - an anvilDraw an quarter - an anvil

• The catalytic cycle of an enzyme

Substrates

Products

Enzyme

Enzyme-substratecomplex

1 Substrates enter active site; enzymechanges shape so its active siteembraces the substrates (induced fit).

2 Substrates held inactive site by weakinteractions, such ashydrogen bonds andionic bonds.

3 Active site (and R groups ofits amino acids) can lower EA

and speed up a reaction by• acting as a template for substrate orientation,• stressing the substrates and stabilizing the transition state,• providing a favorable microenvironment,• participating directly in the catalytic reaction.

4 Substrates are Converted intoProducts.

5 Products areReleased.

6 Active siteIs available fortwo new substrateMole.

Figure 8.17

3b. Chemical Bond Strain3b. Chemical Bond Straintease the bond to fall apart tease the bond to fall apart

Chemical Bond StrainChemical Bond Strain

Stabilize a Stabilize a FictitiousFictitious

statestate

CofactorsCofactors

• CofactorsCofactors– Are nonprotein enzyme helpers, eg

Zn++

• CoenzymesCoenzymes– Are organic cofactors

Non-polypeptide things at the active siteNon-polypeptide things at the active sitethat help enzymes do their jobthat help enzymes do their job

4. Enzymes “partake” in reactions but are not consumed in them

Converts MANY “A’s” into “B’s”

H+

OH-

H+

Partakes: but start and end with the same enzyme configPartakes: but start and end with the same enzyme config

Lysozyme

Lysozyme: kills bacteriaWorks at pH 4-5 Why?

Enzymes:

1. Bring reactants (substrates) in close proximity

2. Align substrates in proper orientation

3. Can act as a Lever: a press or an anvilsmall shape change translates to large force

4. Release products when reaction donerebind more substrates

5. Many small stepsMany small steps, each easily achievedrather than one huge leap

SUMMARY

You expect me to

JUMP this?

No ProblemDude

Enzymes carry out reactions in a series ofsmall steps rather than one energetic event

Reaction rates:

Example: H2O2-> H2O +O2

uncatalyzed –months

Fe+++ 30,000x faster

Catalase 100,000,000 x faster

Enzyme kinetics- kinetikos – moving

Rateor

velocityvelocity

# madeper min

Substrate Conc

maximum velocityVmax

1/2 V1/2 Vmaxmax

KKmm

“substrate affinity”

An enzyme catalyzed rxnAn enzyme catalyzed rxnCan be “saturated”Can be “saturated”

The lower the Klower the Kmm the betterthe enzyme recognizes substrate “finds it at low conc”

The higher the Vhigher the Vmaxmax the more substrate an enzyme can process per min (if substrate around)

“top speed”

“mpg”

Things that affect protein structureThings that affect protein structure often affect enzyme activityoften affect enzyme activity

temperature

pH

pH0 1 2 3 4 5 6 7 8 9 10

0 20 40 60 80 100 º C

Enzyme regulation:Enzyme regulation:

Activity controlled

Continually adjusted

Principal Ways of Regulating EnzymesPrincipal Ways of Regulating Enzymes

Competitive Competitive InhibitionInhibition

AllostericAllosteric Inhibition Inhibition

Covalent Modification (Covalent Modification (phosphorylationphosphorylation) )

-

HO

OH

HO

OH

+

HO

OH

HO

OH

CompetitiveInhibitors:

bind to active site “unproductively”and block true substrates’access

I

S1S2

S & I bind to same site

Competitive inhibition

Allosteric Inhibitors“other” “site”

Distorts the conformation of the enzyme

Negativeallosteric regulator

Allosteric inhibition

Positive Positive allosteric regulators

Helps enzyme work betterpromotes/stabilizes an “active” conformation

Allosteric activation

Allosteric regulators Allosteric regulators change the shapechange the shapeconformationconformation of the enzyme of the enzyme

Stabilized inactiveform

Allosteric activaterstabilizes active fromAllosteric enyzme

with four subunitsActive site

(one of four)

Regulatorysite (oneof four)

Active formActivator

Stabilized active form

Allosteric activaterstabilizes active form

InhibitorInactive formNon-functionalactivesite

(a) Allosteric activators and inhibitors. In the cell, activators and inhibitors dissociate when at low concentrations. The enzyme can then oscillate again.

Oscillation

Figure 8.20

A frequent regulatory modification of enzymesPhosphorylationPhosphorylation

inactive

+ P active

Phosphorylase kinase

Summary

1.enzymes are catalysts2.Lower activation energy EA

3.Mechanism of action …4.Enzyme kinetics- Vmax, Km

5.Regulation of enzyme activity - competitive, allosteric

phosphorylation