section4 enzymes 11

7/30/2019 Section4 Enzymes 11

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Enzymes

Enzymes

Catalyst: A substance that speeds up the rateof a chemical reaction but is not itselfconsumed.

Most biological catalysts are proteins, enzymes. A few catalysts are RNA: peptide bond

formation is catalyzed by RNA in ribosomes.

Some enzymes require organic coenzymes andor metal ions.

Apoenzyme / Apoprotein = Protein

Holoenzyme = Protein + Coenzyme


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Enzymes

Classification of Enzymes

Add -ase to the activity to obtain the name.

1. Oxidoreductases: transfer e- as H or H-

2. Transferases: transfer groups between molecules

3. Hydrolases: add functional groups to water

4. Lyases: form or add to double bonds.5. Isomerases: isomerize by group transfer

6. Ligases: form C-C, C-S, C-O, C-N bonds,coupled to ATP cleavage

Enzymes

Why are enzymes necessary?

1. Enzymes catalyze chemical reactions. They canacceleratebond formation and breakdown by 106-1012

2. Enzymes are responsible for the majority of all reactionsin living systems.

3. Enzymes are very specific; no side-reactions.4. Enzymes can be regulated.


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Enzymes

Our discussion of enzymes will be organized

into three sections:

1.Thermodynamic background to catalysis2.Mechanisms of catalysis3.Kinetics of catalysis

Enzymes

Fundamental terms and ideas for enzyme catalysis:

a) Substrate (S): the reactant on which the enzyme acts,

to convert it to the product (P)

in many cases, two (or even more) substrates reacttogether; there may be two or more products

b) Enzyme-substrate (E.S) complex: a non-covalent,

reversible association between enzyme and substrate

catalysis occurs in the E.S complexc) Active site: the pocket on the enzyme where the

substrate binds and the reaction is carried out


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Enzymes

(same structure; two representations)

Enzymes

We formulate the uncatalyzed reaction as:

S S P

and the enzyme-catalyzed reaction as:

S + E E.S E.S E.P E + P

Here, the double dagger symbol () refers to an activated

state ofincreased energy (the transition state), which

reactants must pass through on their way to the products. E.S and E.P are the non-covalent complexes

between enzyme and substrate or product.


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Enzymes

Transition state theory is a useful way to analyze what is

needed for catalysis to occur.

The rate of a chemical reaction depends on how muchenergy the reactant (or substrate) must acquire in order to

reach the transition state. In general:

reaction rate = (constant)T e -G /RT

Here, G is the activation energy, the extra energy that

S must acquire to reach the transition state S.

S is of higher energy than S because S mustundergo distortion (bond stretching or bending, locking in a

rare conformation) in order to react

Enzymes


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Enzymes

Since the enzyme catalyzes the reaction (makes it gofaster), it must somehow reduce the activation energy.

The situation for uncatalyzed and enzyme-catalyzed

reactions is shown below:

Enzymes

How does the enzyme do this? A key insight is obtained

from the diagram for the catalyzed reaction, where the

complexes E.S and E.P are intermediates:


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Enzymes

The E.S complex is partway along the reactioncoordinate, the horizontal dimension that measures how

far S has progressed in its conversion to P

that is, in the E.S complex S is partly distorted so it

starts to look like the transition state, S

For the uncatalyzed

reaction, S could not

move this far without

an input of free energy(see red arrow)

Enzymes

When S is bound to enzyme, no extra energy seems to be

needed to reach the distorted form seen in the E.S

complex. How could this be you ask?

1. Evidently, the formation of the E.S complex isenergy-releasing (spontaneous, favourable)

2. Some of this binding energy is used to distort thesubstrate, moving it along the reaction coordinate

3. Thus, even in the ground state E.S complex, theenzyme is already working to catalyze the reaction


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Enzymes

Enzymes

The source of energy which results in the lowering ofG is thebinding ofE to S in which a number of weak interactions areformed: H-bonds, hydrophobic interactions, van der Waalsinteractions.

The energy gained in this way (perhaps ~ 50-100 kJ/mol), wouldaccount for how an enzyme can speed up a reaction >1000x.

Other effects of E.S formation include:

1.A decrease in entropy of S (only one conformation)2.Desolvation (removal of H2O shell around S)3.Induced fit: the enzyme adjusts to the shape of S (the transition

state)

4.Alignment of the groups that must react


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Enzymes

How does the enzyme recognize & bind a substrate?

Probably the most important determinants are:

1. Shape consistency or fit (lock and key thesubstrate must fit the active site)

2. Electrostatic consistency - correct matching ofionic and H-bonds within the active site

3. Thermodynamic consistency - can the proteinflex to adopt a substrate or thesubstrate flex to fitinto the active site?

Enzymes

Thus, enzymes provide a special environment inwhich bond formation / breakage is easier.

Example: consider the hydration of carbon dioxide toproduce bicarbonate

H2O + CO

2HCO

3

-H+

+ In the absence of an enzyme, the above reaction is

slow because energy is required to break the O-H

bond of water and stretch one of the C=O bonds.


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Enzymes

As before, we can keep track of the free energy (G)changes by a reaction co-ordinate diagram.

H2O + CO

2HCO

3

-H+

+

P

S

G

Reaction co-ordinate

Lots of G must be added

to stretch the bonds to the

point of breaking in order

to drive the above

reaction.

Enzymes

The reaction catalyzed by Carbonic Anhydrase:

P

S

G

Reaction co-ordinate

E CO2

H2O E +E HCO3

-+ H

+HCO

3

-

is 106 times faster than

the uncatalyzed reaction!

Note that Keq is not affected by

a catalyst. If the enzyme

increases the forward rate k1,

then the back rate k2 will also

be increased since

G P S = G Gp

is lower as well.


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Enzymes

Binding Energy is also used for:1. Entropy Reduction: hold the substrates close

together in proper orientation for reaction.

C

C

O

O

OR

O-

C

C

O

O

O

A

+ OR-

B

CH3C OR

O

CH3C O

-

O

+

CH3

C

O

O

O

CCH3

+ OR-

Fore.g. reaction

A is 105 times

faster than

reaction B.

Enzymes

Binding Energy is also used for:

2. Desolvation: Substrate molecules are surroundedby a water hydration shell that must be removed forreactions to occur.

3. Strain Reduction: Steric and/or electronic strainmust be overcome.


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Enzymes

Enzyme Specificity: Generally, the enzyme is large compared to the substrate and

the substrate binds at a unique location, the active site.

Also, generally, each enzyme catalyzes one reaction using alimited number of substrates.

This enzyme will recognize only D-amino acids, and aseparate enzyme is needed to recognize L-amino acids.

e.g. 1) optical (chiral) specificity

C NH3+

H

COO-

CH2

OH

C

COO-

O

CH2OH

D-serine hydroxypyruvate

D-aminoacid

oxidase

Enzymes

Enzyme Specificity:

eg. 2) geometric specificity

CHOOC

C

COOH-

--

-

CHOOC

H

C

COOH

HO

+ H2O

Fumarate Malate

Fumarase converts fumarate (trans isomer) to malate,

but cannot use maleate (cis isomer) substrate

recognition is very specific.

-

-

C

H COO

C

COOH

Maleate

Fumarase


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Enzymes

Enzyme Specificity:

eg. 3) diastereomeric specificity

COOH

CH2

C COOHHO

CH2

COOH

COOH

CH2

C COOHH

C

COOH

HHO

Citrate Isocitrate

Aconitase

Aconitase can distinguish between the two ends of

citrate even though there is no chiral C - how??

Enzymes

Enzyme Specificity:

eg. 3) diastereomeric specificity

The enzyme provides a binding site that is complementary to the

steric and electronic features of the substrate: hand-in-glove

It can, because the

enzyme is a 3-

dimensional molecule

with 3 sites of

interaction:

O-

O

CCH2

CC

OHH

C

H O

O-

CO

O-

A B

C


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Enzymes

Mechanisms of enzyme catalysisEnzymes use their AA side-chains to participatein the chemical transformation. Here is a classicexample:

General Acid-Base Catalysis

Recall that chymotrypsin is a proteolytic enzymethat cleaves peptide bonds at Trp, Tyr, Phe.

There two important types of catalytic amino acidsin the active site:Acidic residues: donate H+ & accept electrons

Basic residues: accept H+ & donate electrons

Enzymes Mechanism of chymotrypsin

3D Structure of chymotrypsin highlighting

the active site of the enzyme.

catalytic triad

aromatic side

chain pocket


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Oxyanion hole

Aromatic side

chain



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Enzymes

The side chains ofHis57, Asp102, Ser195 in

chymotrypsin form a Catalytic Triad.

Mechanism of chymotrypsin

1

Enzymes

ES complex

The next step involves

electron flow (arrows)

from the General Base

Catalytic Triad into S.

This creates the 1st

tetrahedral intermediate

(next slide).


Substrate binding

compresses the bondbetween Asp102 and

His57, altering the pKa

of His57 to >12.

2


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Enzymes

1st Tetrahedral

Intermediate

The oxyanion isstabilized by H-bonding

to groups in the protein

known as the oxyanion

hole. Notice that a

covalent bond has formed

between the E (Ser195)

and S.

Next, electrons flow from thesubstrate to the General Acid

Catalytic Triad (arrows).


3

Enzymes

Acyl-enzyme

intermediate

The C-N bond has now

been cleaved and the C-

terminal peptide

(stabilized by the

donated proton) is

released:

Note: Acyl group:


4


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Enzymes

Acyl-enzyme

intermediate


The N-terminus is now

going to be released by

hydrolysis:

An incoming water molecule

is activated by His57 (acting

as ageneral base) to create a

hydroxyl group that attacks

the carbonyl carbon of the

enzyme-substrateintermediate

5

Enzymes

2nd tetrahedral

intermediate


Next, His57 acts as a general

acid, protonating Ser195,

causing collapse of the 2nd

tetrahedral intermediate.

6


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EP complex


The N-terminal product is

now free to depart from

the active site and thecatalytic triad is ready to

receive another substrate

molecule.

7


NOTE: The latter half of the

reaction is the same as in the

first half: General base followed

by general acid catalysis.


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Enzymes

Why on earth study protease mechanism (and many

other enzymes) to such a degree?

Answer: These mechanistic studies provide

fundamental knowledge for understanding the molecular

basis of life, modern drug development, medicine and

agriculture!

Enzymes and HIV

HIV RNA genome

Translation

HIV polyprotein

HIV proteinase

Virus assembly

Individual (now active)

HIV enzymes and

proteins


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Enzymes

X-ray crystal structure of HIVproteinase bound to the

inhibitor Viracept.

Kaldor S.W. et al. (1997)J. Med. Chem. 40, 3979-3985.

HIV proteinase inhibitors are

designed using a combination of

structure based drug design

and enzymology research

Enzymes

Mechanisms of enzyme catalysis

About 1/3 of all enzymes use metal cofactors.1. Weak interactions between metals and the substrate help

stabilize charged transition states and may help orient andbind the substrate.

2. Metals accept & donate electrons in Redox reactions


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Enzymes

Mechanisms of enzyme catalysis

Carboxypeptidase uses zinc

in its active site as an

alternative to using amino

acids to form an oxianion

hole (chymotrypsin).

Note the proximity of

reactive sites and the

importance of binding

interactions

Enzymes

3. Enzyme kineticsBy way of review, for the reaction S P:

Every enzyme will have an optimum set of conditions including

temperature, [salt], pH etc. which must be determined experimentally.

V =d[P]

dt=

d[S]

dt

In moles per L per sec.


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Enzymes

Once the optimal conditions are determined, thesestandard conditions are employed in the main goal of

enzyme kinetics: The determination of v [S]or

the influence of[S]on the velocity which is related tothe enzyme-substrate affinity.

The hyperbolic relationship

between v and [S] is typical

of an enzyme that exhibits

what are called Michaelis-Mentenkinetics

Enzymes

Deriving the M-M equation is relatively easy andillustrates some of the principles underlying enzyme

kinetics. Consider the following mechanism:

Assumptions include:

1. k3>>k4 so k4 is not a factor (which is the caseearly in the reaction when [P] = 0).2. k1>k3 which makes E + P formation the slow orrate determining part of the reaction.

i.e. v = k3[ES] defines the overall velocity

E + S E.S E + P

k1

k2

k3

k4


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Enzymes

Enzyme can exist free and substrate bound:[Etotal] = [E] + [ES] (eqn A)

The maximum velocity VMAX will occur when allE isbound to S or,

[Etotal] = [ES] or VMAX = k3[Etotal]

E + S E.S E + P

k1

k2

k3

k4

Enzymes

Derivation of the Michaelis-Menten equation

Rate of formation of E.S = k1[E][S]

Rate of breakdown of E.S = k2[E.S] + k3[E.S]

In steady state (when [E.S] remains constant)k1[E][S] = (k2+k3)[E.S] or,

[ES] =k1[E][S]

(k2 +k3)=

[E][S]

(k2 +k3

k1)

E + S E.S E + P

k1

k2

k3

k4


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Enzymes

Derivation of the Michaelis-Menten equation[ES] =

k1[E][S]

(k2

+k3)

=[E][S]

(k

2+k

3

k1

)

we can define:

KM=

k2+ k

3

k1

(the Michaelis constant)

[ES] =[E][S]

KM

So, (eqn B)

Using [Etotal] = [E] + [E.S] we get: [E] = [Etotal] - [E.S]

Substitute this into eqn B: [ES] =([E

total] [ES])[S]

KM

From

Enzymes


[ES]=([E

total] [ES])[S]

KM

is now rearranged:


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Enzymes


Remember also thatVMAX = k3[Etotal] or,

From above,v = k3[ES] andwe can insert eqn C to give:

V =k3[E

total][S]

KM+ [S]

V =VMAX

[S]

KM + [S]

Michaelis-Menten Equation

Enzymes

The equation is straightforward but the significance and meaning of the KM is

very important:

1. KM is a measure of the affinity of E for SK

M=

k2+ k

3

k1

And since k2>>k3 very often,

KM k2

k1Keq

1 for

Thus, a high KM reflects fast dissociation or a low affinity. A low KM reflects slow

or limited dissociation or a high affinity.

E + S E.S

k1

k2

E + S E.S E + P

k1

k2

k3

k4


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Enzymes

2. KM = [S] at 1/2 VMAX For example,

-Thus, a high KM reflects the need for high [S] for areaction (ie an unfavorable reaction).

-A low KM reflects the need for a low [S] for a reaction(ie a favorable reaction).

3. It follows then when KM = [S], 1/2 of E is

bound to S or [E.S] = [E]

Enzymes

KM values are typically in the M to mM region

Determination of KMAttempts to calculate KM from a v vs. [S] graph is

complicated by the necessity of estimating VMAX

The Lineweaver-Burk plot

can give a more accurate

determination (not used

anymore in practice, butprovides a visual means to

determine KM and VMAX)

Vo

Vmax

[S]

Vmax

2

Km


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Enzymes

The Lineweaver-Burk plot equation:

OR

This is an equation for a straight line (y=mx+c)!

Simplifies to:

Enzymes

The Lineweaver-Burk plot: Note: Because you neverreach [S] = , VMAX is

never reached, but

extrapolation of a straight

line is possible


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Enzymes

Other kinetic constants

kcat (catalytic rate constant) = k3Reflects the slow step and the turnover rate of the enzyme.

That is, v = k3[ES]

and remember VMAX = k3[Etotal]

or VMAX = kcat[Etotal]

Therefore,kcat =

VMAX

[E total]

with units t-1(1/time)

That is, how many times per seconddoes a reaction take

place. Kcat values range from 5x10-1 to 4x107 per sec.

E + S E.S E + P

k1

k2

k3

k4

Enzymes

Other kinetic constants

kcat

KM

The specificity constant

(Reflects the catalytic efficiency of an enzyme)

v VMAX

[S]

KM

(when KM >> [S])

v =

kcat

KM

[Etotal

][S]

It is used to compare catalytic efficiencies of different

enzymes or turnover of different substrates. It is much more

accurate than using one of the parameters on its own.


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Enzymes

Enzyme inhibitors - are categorized on the basis ofhow they affect enzyme kinetic parameters.

An inhibitor is a compound which when added to a

solution containing enzyme and substrate reduces the

rate of conversion of S P.

1.Competitive2.Noncompetitive orMixed3.Uncompetitive

There are three main types of reversible inhibition:

Enzymes

1.Competitive Inhibitors - resemble the substrate and bindin the active site, blocking access of the natural substrate. e.g

Lipitor, Viagra, Protease inhibitors, AZT

E + S + I ES + EI


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Enzymes

1.Competitive Inhibitorse.gmalonic acid, is an inhibitor of succinate dehydrogenase:

COOH

CH2

CH2

COOH

COOH

C

C

HOOC H

H2H

Succinic Acid Fumaric Acid

NOTE: At high [S] the I is displaced from E so Vmax is unchanged.

Enzymes

1.Competitive InhibitorsBut Km is apparently increased because it takes much more S

to reach Vmax. Recall that Km is the apparent affinity of E for S.

Vo

Vmax

[S]

Vmax

2

Km KmI

I


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Enzymes

1.Competitive InhibitorsBut Km is apparently increased because it takes much more S to

reach Vmax. Recall that Km is the apparent affinity of E for S.

1Vo

1 / [S]

I

-1/Km -1/KmI

1

Vmax

Enzymes

2. Non-Competitive Inhibitors

I binds at a site distinctfrom the substrate site, usually

an allosteric site. Allos - Greek - other Stereos -

shape

S

EI

It may bind tofree Eor to E.S. Once bound it will prevent

P formation.


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Enzymes


S

EI

ESE + S E + P

+ +

I I

EI + S EIS X

KIKI

If the binding affinities ofI to E and ES are identical, therewill be no effect on Km.

But since the I decreases active [Etot] then Vmax must

decrease (Vmax = kcat [Etot] ).

Enzymes


If the binding affinities ofI to E and ES are identical, therewill be no effect on Km.

But since the I decreases active [Etot] then Vmax must

decrease (= kcat [Etot] ).

[S]Km

Vmax

I

Vo

VImax

VImax

2


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Enzymes


1 / [S]

1Vo

-1/Km

I

1/Vmax

1/VmaxI

(If the affinity ofI for E and ES are different, Mixed

Inhibition is obtained and both Vmax and Km are changed.)

Enzymes

3. Uncompetitive Inhibitors eg. Roundup

Again, I binds at an allosteric site, but only to the ES complex.

The slopes of 1/Vovs. 1/[S] are unchanged but Vmax is lower, and

so is the apparent [S] needed to reach 1/2 Vmax = Km.


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Enzymes

3. Uncompetitive Inhibitors


The slopes of 1/Vovs. 1/[S] are unchanged but Vmax is lower, and

so is the apparent [S] needed to reach 1/2 Vmax = Km.

VImaxVo I

Vmax

Km [S]KIm

Enzymes

3. Uncompetitive Inhibitors


The slopes of 1/Vovs. 1/[S] are unchanged but Vmax is lower,

and so is the apparent [S] needed to reach 1/2 Vmax = Km.

Vmax

1

-1/KmI -1/Km

I

1 / [S]

1Vo

1

VmaxI


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Enzymes

Allosteric Enzymes vovs [S] plot is Sigmoidal whereas M-M plots are

Hyperbolic.

[S]

Vo

Sigmoidal

Hyperbolic

The enzyme will be very sensitive to [S] over a

narrow range and behaves like an on-off switch.

Enzymes

Allosteric Enzymes The non-MM behaviour arises in multisubunit

enzymes where the occupancy of an active site on

one subunit has an effect on the other subunits.

This is called cooperativity.

The binding of S to one active site of the enzymemakes the binding of subsequent S easier. How?

First, each subunit can exist in 2 conformations:

High affinity - R-state (relaxed)

Low affinity - T-state (taut)


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Enzymes

Allosteric Enzymes Without S, the equilibrium favours T and weak binding.

Binding of S stabilizes the R-state pulling the equilibrium

to the high affinity form simply by the Law of Mass

Action:

+ SS

S

T R

Lots Little

S

S

S

Increasing the amount ofR-state creates

more high-affinity sites and results in more Sbinding, until the enzyme is saturated.

This can be shown to produce sigmoidal Vo

vs. [S] graphs.

Enzymes

Allosteric Enzymes Anything that displaces the equilibrium will affect the

kinetic curveand this includes activators (A) &

inhibitors (I).

Inhibitors bind selectively to T-state, and therefore pullthe equilibrium towards T-state. Activators bind to R-

state (as S does) and pulls the equilibrium towards R-

state:

+I +A AI


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Enzymes

Allosteric EnzymesFor example, the enzyme phosphofructokinase(substrate: fructose-6-P) is inhibited by high levels ofATP and activated by AMP.

Vo+AMP

+ATP

[Fructose-6 phosphate]

Inhibitors and activators do

not bind at the activesite,

but at otherallosteric sites,

from which they can still

influence the R T

equilibrium of the protein.

Enzymes

Allosteric proteins Hemoglobin is not an enzyme, but allostery is an important part

of its function. Hemoglobin binds O2 cooperatively. This allows

it to respond to changes in O2 demand by different tissues.

HMS Cell Biology Visualization website


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Enzymes

Allosteric proteinsO2 binding to hemoglobin is also inhibited by 2,3-bisphosphoglycerate (BPG),

always present in red blood cells. Because fetal hemoglobin (Hb F) has a lower

affinity for BPG than adult hemoglobin (Hb A) does, it has a higher affinity for

O2. This situation permits the fetus to extract O2 from mothers blood.

Enzymes

Advantages of Allosteric enzymes A metabolic pathway is one in which the product of

the 1st Enzyme is a substrate for the 2nd Enzyme etc.

Usually regulation occurs at the beginning of thepath to prevent waste.

In this example, E1 is threonine dehydratase. It isthe key regulatory enzyme in the pathway and is

inhibited by the end product,L-Ile.

E5

E4

E3E2E1

DCBA L-IleL-Thr


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Enzymes

Advantages of Allosteric enzymes

This is known as end-product inhibition orfeedback

inhibition

end-product inhibitors bind at allosteric sites, since

they do not resemble the substrate for the firstenzyme, and cannot bind at the active sites.

E5

E4

E3E2E1

DCBA L-IleL-Thr

Enzymes

Advantages of Allosteric enzymesA more subtle advantage lies in increased sensitivity to

changes in [S] over a narrow [S] range:

Over a physiological substrate range, activity of the

allosteric enzyme is much more sensitive to [S] than the

MM enzyme is, allowing tight regulation of its activity.


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Enzymes

Other types of regulationCovalent Regulation

e.g. Glycogen phosphorylase is activated by phosphorylationof Ser (catalyzed by a protein kinase) a covalent

modification that induces an allosteric conformational

change. This is reversible by a phosphatase that removes

the phosphate from the Ser side chain.

Phosphorylation of enzymes to activate them is an importantmetabolic control mechanism.

A number of other covalent modifications of enzymes arealso known.

Enzymes

Other types of regulation

Zymogens Enzyme activation by proteolytic cleavage. The pancreas produces inactive trypsinogen,

chymotrypsinogen, proelastase, procarboxy-peptidase to

prevent digestion of the pancreas.

A duodenal enzyme enteropeptidase activates trypsin byremoving AA 1-6 of trypsinogen

Trypsin then activates the other proenzymes, whichfunction in digestion of proteins in the duodenum


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Enzymes

Irreversible Enzyme Inhibitors An irreversible inhibitor forms a covalent bondwith an

active site AA.

Penicillin reacts with the active site Ser in transpeptidase,an enzyme involved in bacterial cell wall synthesis.

Aspirin acetylates Ser in the enzyme COX-2 (producesprostaglandin, a hormone that stimulates inflammation),

and relieves inflammation-linked disease like arthritis.

Diisopropylfluorophosphate reacts with Ser-195 inchymotrypsin, inactivating the enzyme.

Enzymes

Summary of enzymes Energetics of catalysis Enzyme specificity Mechanisms of enzyme catalysis Enzyme Kinetics - Michaelis-Menten eqn

KM, kcat, Lineweaver-Burk plot Inhibitors - competitive, uncompetitive,

noncompetitive Allosteric enzymes - equilibrium RT Other types of regulation

section4 enzymes 11

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