asy enzymes 08

8/8/2019 ASY Enzymes 08

1/102

06.12.2008 ASY/Enzymes 1

ENZYMES

Prof.Dr. A. Sha Yaln


2/102


Enzymes are highly specialized

proteins, they have evolved tocatalyze reactions in biological

systems and organisms.


3/102


History of enzymes

1800s........... Digestion of meat by secretions of the

stomach.1850s........... Fermentation of sugar into alcohol by

yeast.

1850s........... Yeast extracts ferment sugar to alcohol.1926............ Isolation and crystallization of urease.

1930s........... Crystallization of pepsin and trypsin

Today...........Nearly two thousand different enzymesidentified, hundreds have been crystallized.


4/102


Properties

Extraordinary catalytic power

High degree of specificity for substratesNo by-product formation

Function under mild conditions of temperature and

pH


5/102


All enzymes are proteins (except for some

catalytic RNA molecules)The primary, secondary, tertiary and

quaternary structures of enzymes are all

essential to their catalytic activity

MW of enzymes range from 12,000 to >

1,000,000


6/102


Some enzymes require an additional

chemical component other than their aminoacid residues for activity. Such additionalgroups are called cofactors

Cofactor(s) may be

one or more inorganic ions

a complex organic or metallo-organic molecule


7/102


Fe2+

Cu2+

Zn2+

Mg2+

Mn2+

K+

Ni2+

Cytochrome oxidase

PeroxidaseDNA polymerase

Hexokinase

Arginase

Pyruvate kinase

Urease


8/102


TPP

FADNAD

CoAPP

Co-B12THF

Transfer of aldehydes

Transfer of hydrogen atomsTransfer of hydride ion

Transfer of acyl groupsTransfer of amino groups

Transfer of H / alkyl groupsTransfer of 1 C atoms


9/102


Prosthetic group covalently bound organic

molecule or metal ion

Coenzyme (Cosubstrate) tightly but notcovalently bound organic molecule


10/102


A complete, catalytically active enzyme together

with its coenzyme and/or metal ions is called aholoenzyme.

The protein part of an enzyme is called the

apoenzyme or apoprotein.

HoloenzymeApo


11/102


Enzyme Names

End in ase

Identifies substratesucrase reacts with sucrose

lipase - reacts with lipid

Describes function of enzyme

oxidase catalyzes oxidation

hydrolase catalyzes hydrolysis

Common names are still used

Pepsin, trypsin


12/102


1. Oxidoreductases Transfer of electrons

2. Transferases Group-transfer reactions

3. Hydrolases Hydrolysis reactions

4. Lyases Addition to double bonds5. Isomerases Group-transfer in molecules

6. Ligases Bond forming reactions (ATP)

Classification


13/102


ATP + Glucose ADP + Glucose-P

Trivial name: Hexokinase

Systematic name: ATP : glucose phosphotransferase

Classification no.: EC 2.7.1.12: Class name (transferase)

7: Subclass (phosphotransferase)

1: Sub-subclass (hydroxyl group as acceptor)1: Glucose as phosphate group acceptor


14/102


Under biologically relevant conditions,

uncatalyzed reactions tend to be slow.

Enzymes provide a specific

environment within which a givenreaction is energetically more favorable.

Enzymes and catalysis


15/102


Active siteEnzymes are proteins that catalyze chemical reactions.

Folding of the protein into itstertiary structure brings side-

chains of various amino acidsthat may be far apart in theprimary sequence into close

juxtaposition, forming anactive site.


16/102


Enzyme

Active site

SWater P


17/102


Reactive groups at the active site catalyze reactions by:

donating or withdrawing electrons

stabilizing or generating free radical intermediates

forming temporary covalent bonds (a transition state

intermediate)

There is high degree of specificity for the reaction catalyzed,i.e. an amino acid bound to pyridoxal phosphate may undergo:

isomerization, decarboxylation, transamination or side-

chain eliminationbut an enzyme will normally catalyze one of these reactions.


18/102


1. Amino acids at the active site will make non-covalent interactions

between their side-chains and substrate molecule(s):

acidic groups (Asp, Glu) basic groups (Lys, His, Arg)

hydrophilic interactions with OH groups (Ser, Thr, Tyr)

hydrophilic interactions with SH groups (Cys) hydrophilic interactions with amide groups (Asn, Gln)

aromatic interactions (Phe, Tyr, Trp)

hydrophobic interactions (Ala, Leu, Ile, Val, Met, Pro)Amin2. Metal ions, carbohydrates and lipids bound to the enzyme may also

interact with substrates.3. Binding may result in considerable conformational change.


19/102


There is specificity in binding to the active site

Because of multiple interactions in binding to the active site,enzymes can readily distinguish between isomers

C

C

OH

OH

H

CH2OH

C

COO-

CH3

NH3+H

D-glyceraldehyde D-alanine

C

C

H

OH

HO

CH2OH

C

COO-

CH3

H+H3N

L-glyceraldehyde L-alanine


20/102


Enzymes can also distinguish between isomers

cis

trans


21/102


Enzymes act by lowering the activation

energy of the reaction

initialexcited

final

+ enzyme

non-enzymicener

gylevel

They increase the speed at which equilibrium is achieved,but they do not alter the position of the equilibrium.


22/102


Freeenergy

of

system

Progress of reaction

Activation energy barrier

Activation energy

of the catalyzed

reaction

Activationenergy

of the

uncatalyzed

reaction

Initial

state Overall

free-

energy

change

Final state at equilibrium


23/102


Effect of pH

Enzymes have maximum activity at theiroptimum pH

Tertiary structure of enzyme must be maintained

Most enzymes have a narrow range of activity;

they loose their activity at low or high pHR groups of amino acids at the active site

determine pH profile


24/102


Relative

rate

pH

2 4 6 8 10 12

Pepsin Glucose 6-phosphatase


25/102


Effect of temperature

Enzymes will have very little activity at low

temperaturesReaction rate increases with temperature

Enzymes are most active at their optimum

temperatures (usually 37C in humans)

At high temperatures activity will be lost due

to denaturation of protein


26/102


Relative

rate

Temperatureo

C

20 30 40 50 60 70


27/102


Effect of substrate: [S]

Increasing substrate concentration increases

the rate of reaction (at constant enzymeconcentration)

Maximum activity is reached when all theenzyme combines with substrate


28/102


Substrate concentration, M

Initial

rate


29/102


Michaelis-Menten Equation

E + S ES E + P

E: enzyme

S: substrate

P: productES: enzyme-substrate complex

k1

k-1

k2

k-2


30/102


1. Rate of formation of ES

Rate of formation = k1([Et] - [ES]) [S]

2. Rate of breakdown of ES

Rate of breakdown = k-1[ES] + k2[ES]

E + S ES E + P

k1 k2

k-1 k-2


31/102


3. The steady state

Rate of formation = Rate of breakdown

k1([Et] - [ES]) [S] = k-1[ES] + k2[ES]

E + S ES E + P

k1 k2

k-1 k-2


32/102


4. Separation of the rate constants

k1[Et] [S] = (k1 [S] + k-1 + k2) [ES]

[Et] [S]

[ES] =[S] + (k2 + k-1) / k1

E + S ES E + P

k1 k2

k-1 k-2


33/102


5. Definition of initial velocity

vo = k2 [ES]

k2 [Et] [S]

vo =[S] + (k2 + k-1) / k1

E + S ES E + P

k1 k2

k-1 k-2


34/102


6. Definition of Vmax and Km

Vmax = k2 [Et]

Km = (k2 + k-1) / k1

E + S ES E + P

k1 k2

k-1 k-2


35/102


Michaelis-Menten EquationVmax [S]

vo =KM + [S]

vo= initial rate; Vmax= maximum rate;

KM = Michaelis-Menten constant


36/102


Substrate concentration, M

Initial

rate

Vmax

1/2 Vmax

o

o

oo

o

oo

o o o o

KM


37/102


Determination of Km and Vmax

0

0,2

0,4

0,6

0,8

1

0 200 400 600 800

[substrate]

relativeactivity maximum rate of reaction

when the enzyme is saturatedUnits: mol product / time

[Substrate] required to achieve VmaxUnits: mol substrate / L

Vmax

Km


38/102


Lineweaver-Burk plot

1 / [substrate]

1/rate

1 / Vmax

-1 / Km

Slope= Km/ V max


39/102


A high or low value of Km is relative to the physiological

range of concentration of substrate in cells

0

20

40

60

80

100

120

0 100 200 300 400

[substrate], mmol /L

rateofre

action,mol

/min

Km high compared with physiological range of [substrate]

large increase in rate of reactionfor a small increase in [substrate]

Km low compared with physiological range of [substrate]small increase in rate of reaction

for a large increase in [substrate]


40/102


0

20

40

60

80

100

120

0 100 200 300 400

[substrate], mmol /L

rateofreaction,mo

l/min enzyme A

low Km

enzyme Bhigh Km

S

P

X

enzyme A

enzyme B

Two enzymes competing for substrate S


41/102


Bisubstrate reactions

Single-displacement reactions

Double-displacement or ping-pongreactions

E

S1 S2

P


42/102


Single displacement: (A + B C + D)

E + C + D

A

E

B

A

E

BA

E


43/102


E

AX

E

AX

E

E E

B

E

A

BX

BX

Double displacement: (AX + B A + BX)

X

X


44/102


Enzyme Inhibitors

cause loss of catalytic activity

change the protein structure of enzyme

may be competitive or noncompetitivesome are irreversible


45/102


Enzyme inhibitors

Irreversible

Reversible Competitive

Non-competitive


46/102


Enzyme inhibitors

irreversible bind to enzyme covalently

may undergo part of reaction transition state intermediate does not breakdown

reversible non-covalent (equilibrium) binding to enzyme

many are substrate analogues may be relatively unspecific

some inhibitors are used as drugs- mechanism-dependent (suicide) inhibitors

- highly specific for target enzyme- rational drug design


47/102


Irreversible inhibition

E EII

Inactive

enzyme


48/102


Competitive inhibition

E

SES

EII

E-Scomplex

Inactiveenzyme


49/102


Competitive Inhibitors

have a structure similar to substrate

occupy the active site

compete with substrate for the active site

their effect is reversed by increasing

substrate concentration


50/102


[substrate]

rateofreacti

on

1 / [substrate]

1/

rate

ofreacti

on

Vmax unchanged

Km increased

Competitive inhibition


51/102


Noncompetitive Inhibitors

do not have a structure like substrate

bind to the enzyme but not to the active site

change the shape of enzyme and active site so thatsubstrate can not fit altered active site

their effect is not reversed by adding substrate


52/102


Noncompetitive inhibition

ES

ES

E

E-S

complex

Inactiveenzyme

IS

S

I

I


53/102


[substrate]

rateofreactio

n

1 / [substrate]

1/rateofreac

tion

Km unchanged

Vmax

decrease

d

Noncompetitive inhibition


54/102


Uncompetitive inhibition

E

S

ES

E

E-S

complex

Inactive

enzyme S

I

I


55/102


How can we determine whether aninhibitor is reversible or irreversible ?


56/102


dialysis semi-permeable membrane

small moleculesequilibrateacross the membrane

proteins are too largeto cross the membrane


57/102


semi-permeable membranedialysis




58/102





inhibitor removedactivity restored


59/102


dialysis semi-permeable membrane


proteins are too largeto cross the membraneinhibitor boundcovalently to protein


60/102



inhibitor not removedactivity not restored


61/102


Enzymes are measured by their

catalytic activity; not by their mass.


62/102


Factors affecting enzyme activity

pH of incubation or environment

temperature time of incubation

concentration of enzyme

concentration of substrate

covalent modification of enzyme

inhibitors and activators


63/102


Information required for quantitative

enzyme measurement

1. Equation of the reaction

2. Analytical procedure3. Cofactor requirement

4. Substrate concentration dependency

5. Optimum pH6. Temperature dependency


64/102


0

20

40

60

80

100

120

0 100 200 300 400 500 600 700 800

[substrate]

rateo

f

reactio

n

Excess substrate is used so that enzyme is saturated;limiting factor in product formation is [enzyme].


65/102


Determining initial rates at increasing [enzyme]

Time

Progress

of

reaction

Initial

rates

E

3xE

2xE


66/102


Enzyme activity vs. initial rate

Enzyme activity, units

Initial

rate

1

4 6 8 10 122

2

3

4

14

o

o

oo

oo

o


67/102


Enzyme Activity (IU)

One unit of enzyme activity is defined as that

amount causing transformation of 1 mol ofsubstrate per minute under optimal conditions of

measurement.

The specific activity is the number of enzyme units

per milligram of protein.


68/102


H2O

C CN

H

R1

O

N C

H H

R2

C

O

H

COOHH2N

C CN

H

R1

OH

H2N OH N C

H H

R2

C

O

COOH

H

In vitro:10 12 hours in 12 mol /L HCl at 105Crandom hydrolysis of peptide bonds

In vivo:

1 2 hours at 37C, specific bonds hydrolysed

An example of enzyme catalysis: serine proteases


69/102


70/102


Histidine-57CH

HN

C

CH2

O

N N

Aspartate-102CH

HN

C

CH2-OOC

O

Serine-195CH

HN

C

CH2HO

O


71/102



72/102


Bonds hydrolysed:

trypsin

esters of basic amino acids

chymotrypsin

esters of aromatic amino acids

elastase

esters of small neutral amino acids

-

Gly

Gly

Asp

-

+

peptide in groove on enzyme surface

trypsin


73/102


Bonds hydrolysed:

trypsin


chymotrypsin


elastase


Gly

Gly

Ser


chymotrypsin


74/102


Bonds hydrolysed:

trypsin


chymotrypsin


elastase


Val

Thr

Gly


elastase


75/102


Lock-and-key Model

Enzyme binds the substrate in the active site

Only certain substrates can fit the active site

R-groups of amino acids forming the active siteaid substrate binding

Enzyme-substrate complex forms subtratechanges to product product is released fromthe enzyme


76/102


E P2

P1

S S

E E

++

E + S ES E + P


77/102


78/102


E E

E

Unfavorable orientation

Unfavorable proximity

Unfavorable orientation

Favorable proximity

Favorable orientation

Favorable proximity


79/102


S

A

+S

Relaxed enzyme

molecule

Induced fit of enzyme

to the bound substrate

I d d fi d l


80/102


Induced-fit model

Enzyme structure is flexible, not rigid

Enzyme and active site adjust theirshape to bind substrate

Both the enzyme and substrate

undergo conformational changeShape change

increases range of substrate specificityimproves catalysis during reaction


81/102


P2

P1

S S +

E + S ES E + P

E EE+ S


82/102


In the induced fit model;

when substrate binds the shape of the

enzyme adapts to the substrate.

P d /


83/102


Proton donors/acceptors

carboxyl group (-COOH)

amino group (-NH2)

sulfhydryl group (-SH)

imidazole group


84/102


Enzymes act in organized sequences

Some enzymes participating in cellular

metabolism are regulatory enzymes.

E1

A B C D

E2 E3

R l ti f E A ti it


85/102


Regulation of Enzyme Activity

Inhibition

Allosteric regulation Covalent modification

Isoenzymes Synthesis/degradation

All i d l i


86/102


Allosteric modulation

Increased

enzyme

activityM+

Decreased

enzyme

activity

M-

M

M

E

E

E


87/102


v

[S]

+

Normal

rate -


88/102


[substrate]

rateofreaction

Allosteric regulation is instantaneous activation by precursors inhibition by end-products


89/102



90/102

06.12.2008 ASY/Enzymes 90substrate concentration

rateo

freaction

activation due to decreased cooperativity

inhibition due to increased cooperativity


91/102


AA

C

C

A

B

B

S

A

B


92/102


Aspartate Transcarbamoylase

Covalent modification


93/102


Some enzymes are modified by

phosphorylation, glycosylation and otherprocesses.

These alterations effect enzyme activity, and

are involved in the regulation of enzymes.

Covalent modification


94/102


H3PO4

H2O

ADP, H2O

ATP

serinephosphoserine

CH COHN

CH2

O

P

O

HO OH

CH2OH

CH COHN

CH2OH

CH COHN

serine

Covalent modification of an enzyme is

fast: time course of seconds minutes commonly in response to:

fast acting hormones (peptides, etc)


95/102


CH2CH2

OH OH

CH2CH2

OOP P

Phosphorylase a(inactive)

Phosphorylase b(active)

Multiple forms of enzymes


96/102


Multiple forms of enzymes

Many enzymes occur in more than one

molecular form in the same species, inthe same tissue, or even in the samecell.

Such multiple forms of enzymes arecalled isoenzymes or isozymes. Typical

examples are: lactate dehydrogenase(LDH), creatine kinase (CK)

Isoenzymes


97/102


Isoenzymes

Enzymes catalysing the same reaction,

but differing in structure:

may have different charges at a given pH may have different affinity for substrate

may preferentially catalyse reaction in one direction

may differ in temperature sensitivity

may differ in inhibitor sensitivity

may differ in coenzyme specificity

They may be found in different tissues and in different

organelles

Lactate dehydogenase


98/102


Lactate dehydogenase

CH3

C

COOH

O

CH3

CHOH

COOH

NAD+

NADH

NAD+

NADH

pyruvate lactate

pyruvate reduction in skeletal muscle

lactate oxidation in heart muscle

type 1 type 2 type 3 type 4 type 5

Separation of LDH isoenzymes


99/102


Separation of LDH isoenzymes

The different isoenzymes have different charges,and can be separated by electrophoresis.

Type

1

Type2

Type3

Type4

Type5

Physiological control of enzyme activity


100/102

06.12.2008 ASY/Enzymes 100

Physiological control of enzyme activity

Change in the rate of synthesis of the enzyme

(change in gene expression) slow: time course of hours or days

commonly in response to:

slow acting hormones (steroids)

long-term adaptation

Use of enzymes in medicine


101/102

06.12.2008 ASY/Enzymes 101

Use of enzymes in medicine

measurement of metabolites in plasma and urine

measurement of enzymes in plasma

assessment of tissue damage

physiological control of enzyme activity

use of enzyme inhibitors as drugs

Animations


102/102

Animations

http://www.kscience.co.uk/animations/model.swf

http://www.northland.cc.mn.us/biology/biology1111/

animations/enzyme.swf

http://cble.chem.uu.nl/biolip/SERPROTE.SWF

http://www.stolaf.edu/people/giannini/flashanimat/enzymes/allosteric.swf
http://www.kscience.co.uk/animations/model.swfhttp://www.northland.cc.mn.us/biology/biology1111/animations/enzyme.swfhttp://www.northland.cc.mn.us/biology/biology1111/animations/enzyme.swfhttp://cble.chem.uu.nl/biolip/SERPROTE.SWFhttp://www.stolaf.edu/people/giannini/flashanimat/enzymes/allosteric.swfhttp://www.stolaf.edu/people/giannini/flashanimat/enzymes/allosteric.swfhttp://www.stolaf.edu/people/giannini/flashanimat/enzymes/allosteric.swfhttp://www.stolaf.edu/people/giannini/flashanimat/enzymes/allosteric.swfhttp://cble.chem.uu.nl/biolip/SERPROTE.SWFhttp://www.northland.cc.mn.us/biology/biology1111/animations/enzyme.swfhttp://www.northland.cc.mn.us/biology/biology1111/animations/enzyme.swfhttp://www.kscience.co.uk/animations/model.swf

asy enzymes 08

Documents