bioscience unit 2 enzymes

7/25/2019 Bioscience Unit 2 Enzymes

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Enzymes

Enzymes are the catalysts of nature.

In the vast majority of cases, these are proteins.

In some cases, the protein may be "enhanced" by the

addition of a prosthetic group (cofactor), which widens

the catalytic possibilities for enzymes.


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Stereochemistry Note that the R group means that the -carbon is a

chiral center.

All natural amino acids are L-amino acids.

This means that almost all have the Sconfiguration.(Exceptions: glycine and cysteinecan you tell why?)


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Enzymes Like all catalysts, enzymes act by lowering the activation

energy for a specific reaction.

Each enzyme is very specific in terms of the reactants that it

binds, and the reaction that it catalyzes with them.

The reaction can be considered to take place on the surface

of the enzyme.

The equilibrium of the catalyzed reaction is still determined

by the free energy change.


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The M ain Enzyme c lasses

1. O xidoreductases O xidation-reduction reactions

2. Tran sferases Tran sfer of functional groups

3. H ydrolases H ydrolysis reactions

4. Lyases Group elimination (forming new bonds)

5. Isomerases Isomerization reactions

6. Ligases Bond formation coupled w ith

atriphosphate cleavage


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Constitution of the four-digit EC num ber.

EC num ber: E C (i). (ii). (iii). (iv)

(i) Th e ma in class, deno tes the type of reaction it catalyze

(ii) Th e subclass, indicates the type of substrate, type of transfered functiona l group s, the nature of

specific bon ds involved in the catalyzed reaction

(iii) Sub -sub class, indicates the nature of substrate or co-subs trate

(iv) An arbitrary unique serial num ber


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Transition states

Transition states are intermediate states between the ground

states of the reactants and products, through which the reaction

must pass.

Formally, we consider a single transition state at which the

energy is at a maximum (i.e. the least favorable).


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Activation energy

The activation energy (G) is the difference in energy

between the ground state of the reactant(s) and the transition

state.

The larger the G, the less likely the reactants will be at the

transition state, and the slower the reaction will proceed.


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Activation energy

Enzymes catalyze reactions (speed them up) by loweringthis activation energy.

This is because the reaction takes place on the enzyme

surface, where a special environment favorable to thereaction is available.

This is called the active site.

The reactants bound there are called substrates.


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Enzyme Mechanisms

Enzymes carry out their function of catalyzing specific

reactions using a variety of ways.

As you will see, common themes will recur.

While it is relatively easy to propose a mechanism, testingthe hypothesis is not always straightforward and is never

easy.


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General principles used by enzymes:

1) The proximity effect ("entropy reduction"): Enzymes that bind 2 substrates bring both reactants in close

proximity (and usually in the correct orientation for a

productive reaction).

A bimolecular reaction becomes essentially unimolecular,

significantly enhancing the rate.

One can think of it as raising the effective concentration of the

2 reactants:

on the surface of the enzyme, the effective concentration

of a substrate in the vicinity of a second substrate is on theorder of 10 M.

A rate enhancement of ~106 can be achieved for a 10 M

substrate by this effect alone.


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Enzyme act as a molecular template


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Enzyme act as a molecular template


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General principles used by enzymes:2) General acid-base catalysis (GABC):

Many reactions involve the removal and/or addition of protons.

Since most enzymes can only function within a limited pH

regime, they utilize amino acid sidechains as general acids and

general bases to donate or extract protons.

Example: An unprotonated His can act as a general base, and a

protonated His can act as a general acid.


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3) Nucleophilic functional groups and their activation:

Many enzymes form an intermediate state in which

their substrate is covalently linked ("covalent

catalysis").

This temporary state is the result of a nucleophilic attack on

the substrate by a functional group on the enzyme.

(Formation of the acyl-enzyme ester on the active site serine of

serine proteases is an example.)

Often the nucleophilic group is activated by other functional

groups nearby.

Bound metal ions can also serve as electrophiles"metal ion catal sis" .


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General principles used by enzymes:4) Electrostatic effects:

Appropriately placed charged (or partially charged)

groups can stabilize the development of charge in the

transition state.

Electrostatic interactions are long range, but they also

depend upon the dielectric constant of the intervening

medium.

They will be stronger in the interior of a protein than onthe surface.


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General principles used by enzymes:5) Structural flexibility:

This allows enzymes to effectively surround and enclose

their substrates ("induced fit").

The exclusion of water brought about by this changemaximizes the increase in entropy upon binding and

decreases the dielectric constant, thus intensifying the

electrostatic effects.


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The active site Typically a pocket or groove on the surface of

the protein into which the substrate fits.

The specificity of an enzyme fit between the

active site and that of the substrate.

Enzyme changes shape tighter induced fit,

bringing chemical groups in position to catalyze

the reaction.

S b i i


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Substrate orientation

Substrate specificity


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The active site is an enzymescatalytic center

In most cases substrates are held in the active site by

weak interactions

R groups of a few amino acids on the active site

catalyze the conversion of substrate to product.

A single enzyme molecule can catalyze thousands or more

reactions a second.

Enzymes are unaffected by the reaction and are reusable.


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Specificity

Enzymes selectively recognize proper

substrates over other molecules

Specificity is controlled by structuretheunique fit of substrate with enzyme

controls the selectivity for substrate and the

product yield

E b t t ifi


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Enzymes are substrate specific

When a substrate or substrates binds to an enzyme, the

enzyme catalyzes the conversion of the substrate

to the product.

Sucrase is an enzyme that binds to sucrose and

breaks the disaccharide into fructose and glucose.


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6) Binding of the transition state:

One way to do this is to place functional groups such that

they bind the transition state better than the ground state.

The geometry of the transition state is very often

different from the ground state, and this can be used by

the enzyme.


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General principles used by enzymes:6) Binding of the transition state:

The individual interactions that stabilize the binding of the

enzyme to the substrate (transition state) are the same as the

ones that stabilize 3 and 4 structure (H-bonds,

hydrophobic effect, ion pairs, etc.)many weak interactionsadd up to give a significant binding energy.

This is one reason why biological catalysts are so large

they need to be able to provide all of the functional groups(in the proper geometry) for all of these interactions.

This also explains the exquisite specificity of enzymes.


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Serine proteases

Trypsin, chymotrypsin, and elastase form a protein family

with a conserved fold.

Subtilisin is a representative of an independent and

distinct family with a very different structure.


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Serine proteases

All contain a catalytic triad: Asp, His, Ser

Asp: forms a low-barrier H-bond with His

His: serves as base to abstract proton from Ser (or H2O);

donates proton to atom leaving carbonyl C

Ser: makes nucleophilic attack on carbonyl C


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Serine proteases This model explains a lot of data:

Burst kinetics: Serine proteases can also catalyze hydrolysis of

esters. In this case, the first half of the reaction (acylation) is

much faster, and the second half (deacylation) is rate-limiting. In

the pre-steady state phase, 1 equivalent of product forms very

quickly.

pH effects: protonation of His or deprotonation of N-term

(Ile16)

Modification of catalytic triad inhibits activity: DFP

attacks the Ser; His is target of chloromethylketones.

Mutation of any member of triad drastically reduces

activity.


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O

O

14C H

3O

2N

14C H

3 O

Enz

O

N O2

O

14C H

3 N H O H

O

Enz-OH

Radiolabelled enzyme

H2N -O H

Enz-OH (regeneration)

E vidence of an acyl interm ediate form ation


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O HO TS

Serine protease

TS-Cl 0 .2 N OH-

C hem ical m odification of Ser-195 in -chym otrypsin

inactivated enzyme

Inhibition of Serine proteases by affinity labeling


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p y y g


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P

O

F

OP

S

O S

O

O O Et

O Et

O

P

O

O CN

N Me2

P

O

O

F

SarinMalathion

Tabun Soman

Neu rotoxin nerv e gases inhibits acetylcholine esterases (serine-proteases)


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O

O Me

NH

S O2

Me

O

NH

S O2

Me

Cl

N-Tosyl-L-phenylalanine methyl ester,

-chymotrypsin substrate

N-Tosyl-L-phenylalanine chloromethyl ketone,

irreversible inactivator of -chym otrypsin

E vidence for H istidine participation


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E

O H

NH

N

RMe

O

Cl

H E

O H

NH

N

R

Me

O

SN 2

alkylation

Inversion

M ech1: Inactivation of -chymotrypsin by -chlorom ethyl ketones


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E

O H

NH

N

RMe

O

Cl

HE

O

O

R

H

C l

Me

NH

N

E

NH

NMe

O

OR

E

O H

NH

N

R

Me

O

M ech2: Inactivation of -chym otrypsin by -chloromethyl ketones

B: Tetrahedral intermediate formation

SN 2

alkylation

Inversion

Enzyme

regeneration


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E

O H

NH

N

RMe

O

C l

HE

O

O

R

H

C l

Me

NH

N

E

NH

NE

O H

NH

N

R

Me

O

Me

OR

O

M ech3 : Inactivation of -chym otrypsin by -chloromethy l ketones

(double inversion m echanism experimen tally proved)

B: Tetrahedral intermediate formation

SN 2 alkylation

Retention

Intramolecular

Epoxide formation

1st Inversion

Epoxide cleavage

2nd inversion


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AcNHNH

H

O

O

ClP h

(2S)-N-acetyl-L-alanyl-L-phenylalanine- -chloroethane acting as a inactivator, the crystal structure

of the covalent adduct with -chymotrypsin clearly shows that His-57 is alkylated, and the

stereochemistry of the inactivator is retained, supp orting the doub le displacement mechan ism.


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N N H

CO2H

O

ON N H

O

O

NH

N

O

O

Evidence for the d eacetylation mecha nism (G AB catalysis): His participation

Chem ical mod el for the deacetylation step in -chymotrypsin

Good

modearte

unreactive


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N N H

O

O

O

OOH H

NH N

O

O

O HOH O

NH N

O H

O

O H

O

O

Evidence for the deacetylation mechan ism (G AB catalysis): H is participation

Chem ical mod el for the deacetylation step in -chymotryp sin


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Metalloproteases(Exemplified by thermolysin & carboxypeptidase)

Usually use Zn2+ as an electrophile (simple prosthetic

group).

It polarizes the C=O bond, allowing formation of a

tetrahedral intermediate upon attack by OH-, which isgenerated when a nearby carboxylate abstracts a proton from

a bound water molecule.

The water molecule may also serve as a ligand to the Zn2+ion, in addition to the amino acid sidechains provided by the

polypeptide (most commonly His, Glu, Asp, and sometimes

Cys).

Mechanism of carboxypeptidase A


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Enolase


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Enolase

Catalyzes interconversion of 2-phosphoglycerate (2PG) and

phosphoenolpyruvate (PEP)

Uses lyase mechanism:

Lys345 acts as general base to abstract proton from C-2 of

2PG, forming enolate, which is stabilized by strong interactionwith 2 bound Mg2+ ions.

Glu211 acts as general acid, donating proton to C- 3

hydroxyl, allowing it to leave as H2O.


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Lactate dehydrogenase 2-substrate oxidoreductase, which uses a nucleotide coenzyme,

NAD+/NADH.

The coenzyme binds first, driving a conformational change

that allows lactate/pyruvate to bind, positioned properly forthe redox reaction.

Arg171 positions the second substrate via electrostatic

interactions with the carboxylate of lactate/pyruvate.

His195 acts as a proton donor/acceptor to the oxygen bonded

to C-2 (normally H-bonded to it).

LDH-catalyzed reaction


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Low molecular weight component essential forprotein function

Metal ions

Prosthetic groups

Organic / bioorganic e.g.Heme groups

Coenzymes

Cofactors

Apoenzyme + coenzyme Holoenzyme


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Cofactors

not all vitamins are cofactors

all water-soluble vitamins with the exception ofvitamin C are converted/activated to cofactors

only vitamin K of the fat-soluble vitamins isconverted to a cofactor

cofactors may also act as carriers of specificfunctional groups such as methyl groups and acyl

groups

Coenzyme Vitamin Role


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ATP ------------------ Energy and

phosphate

transferNAD(P) Niacin Redox

FAD/FMN Riboflavin (B2) Redox

Coenzyme A Pantothenic

acid (B3)

Acyl transfer

TPP Thiamine (B1) Transfer of 2 C

PLP Pyridoxine (B6) Amino acids

Lipoamide -------------- Acyl transfer

Ubiquinone -------------- Electron carrier

Classes of coenzymes


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Classes of coenzymes

Cosubstrates are altered during the reaction and regenerated byanother enzyme

Prosthetic groups remain bound to the enzyme during the

reaction, and may be covalently or tightly bound to enzyme

Metabolite coenzymes - synthesized from common metabolites

Vitamin-derived coenzymes - derivatives of vitamins (vitamins

cannot be synthesized by mammals, but must be obtained as

nutrients)

bioscience unit 2 enzymes

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