ENZYMES

1

ENZYMESENZYMES

2

Enzymes are proteins

Enzymes are proteins

CofactorsCofactors

Prostetic groups

Metal ions

Coenzymes

Coordinative bond

Covalent bond

Secondary interactions

3

Optimal Conditions:

Ionic strength

Temperature

Solvent

pH

Native conformationNative conformation

Maximal catalytic activity

4

pH dependence of enzyme activitypH dependence of enzyme activity

Active center:

Glu COOH pKa = 5.9

-Lysozyme

Asp COOH pKa = 4.5

Optimal pH?

Example:

pH

4 5 6protonated deprotonated4.5 5.9

Asp - Glu

optimal pH Hw: Glu-, His+ (pKa 6.5)

5

pH

Enzymeactivity

5 64

pH profilespH profiles

pH optimum Isoelectric pont=

6

Temperature dependence of enzyme activityTemperature dependence of enzyme activity

T

Enzymeactivity

Heat denaturationchanges in conformation

Heat sensitivity is different

Heat-shock proteins

Q10 temperature coefficient

Optimal temperature

7

Calculation of enzyme activity:

Enzyme activity (EA) S]EA = t

P]EA = t

U (unit) U = mol/min

S.I. unit: Catal = mol/sec

substrate (S) product (P)

8

Turnover number(Molecular activity)

Carbonic anhydrase: 400 000/sec

mol (product)mol (enzyme) x sec

Chymotrypsin: 100/sec

Trp synthase: 2/sec

examples

9

G

Substrate (S) Product (P)

G

Eact

S ES*1 ES *

2 P ES* : enzyme-substrate complex

S

P

ES*1

ES*2

Enzymes as catalysts

Enzymes as catalysts

10

E.g.: „Stickase”

S PS*

E

ES*1

E

ES*2

11

Effect of enzymes :

The reaction mechanism changes

Activation energy decreases

G (Kes ) does not change

The equilibrium state

The time to reach the EQ decreases

Enzymes are catalizing directionsin both

does not change

12

The enzyme - substrate complex

The enzyme - substrate complex

Interactions:secondary bonds ionic bondscovalent bond (covalent catalysis)

Acid-base catalysis: proton shuttle

13

Enolase and 2-phospho-glycerate (2-PGA): ionic bond

2 Mg2+ cofactors

14

The enzyme-substrate complex (ES)

Enzymes are specific catalysts

Enzymes are specific catalysts

Lock and key model

E

S

„induced fit” model S

E

15

Hexokinase induced-fit

16

Hexokinase induced-fit

17

Entropy effect

Entropy effect

E

S1

S2

Binding of substrates• close in space• proper orientation

18

Dihydrofolate reductase

dihidrofolate

NADP+

cofaktor

19

Dihydrofolate reductase

dihidrofolate

NADP+

cofaktor

20

Medical-diagnostic significance of enzyme activity measurements


1. Non-functional plasma enzymes

accelerated death of certain tissues

soluble enzymes enter the circulation

21

Aminotransferases:a reversible exchange between an -amino and an -keto group

ALAT: alanine-amino transferase (or SGPT: serum glutamate - pyruvate aminotranspherase)

pyruvate alanine

glutamate -ketoglutarate

high ASAT in serum hepatocellular tissue destructione.g. in acute hepatitis (viral infection) in liver chirrosis in mononucleosis

22

ASAT:aspartate-amino transferase (or SGOT: serum glutamate -oxaloacetate aminotranspherase)

oxaloacetate aspartate

glutamate -ketoglutarate

high ALAT in serum muscle tissue destructione.g. in myocardial infarction in acute rheumatoid carditis in the first 10 days of heart surgery after heart massage after catheter treatment of heart

23

Creatine kinaseM: muscle form, B: brain form

MM, MB or BB izoenzymesMB form is found exclusively in the heart

creatine creatine-P

ATP ADP

high MB in serum myocardial diseasee.g. in myocardial infarction

(5-6 hrs after heart attack maximum value at 12 hrs)

colon cancer high MMrenal failure high MM

24



2. Functional plasma enzymes

25

LCAT:lecitine-cholesterol acyl transferase

free cholesterol cholesterol ester

lecitine lysolecitine

Deficiency: cholesterol cannot be transported in the blood

artheriosceloris, heart attack

26



3. Enzyme activity assay in tissue biopsy

27

Pyruvate carboxylase

pyruvate oxaloacetate

ATP, CO2ADP

Deficiency: mental retardation

Assay from skin biopsy

Treatment: oral Asp, Glu

28

Enzymes as Catalysts

Ser Proteases

Enzymes as Catalysts

Ser Proteases

29

Acid-Base CatalysisAcid-Base Catalysis

A general base

His

Cys-S -

Tyr-O -

Asp-, Glu -

A general acid

His+

Cys-SH

Tyr-OH

Lys+

30

Covalent CatalysisCovalent Catalysis

E + S [ES] E + P

Covalent bond in the enzyme-substrate complex

31

Ser ProteasesSer Proteases

Protease: cuts proteinshydrolysis of peptide bond

Ser-proteases: Ser in the active center

e.g.•Chymotrypsin•Trypsin•Ellastase•Thrombin•Other blood coagulation factors

32

DIPF: Specific inhibitor of Ser enzymesDIPF: Specific inhibitor of Ser enzymes

E

OHSer

O

P OF

ODi-isopropyl-fluoro-phosphate

Irreversibleinhibition

of Ser- enzymes

e.g. ChymotripsinAcetylcholinesterase(chemical weapon)

33

Proteolytic activation of zymogensProteolytic activation of zymogens

Intestine Pancreas

Trypsinogen

Chymotrypsinogen

- Chymotrypsin

Trypsin

Enteropeptidase

- Chymotrypsin

Trypsin inhibitorInactive trypsine

34

Interaction of 1NH3+

with 194Asp-

Chymotrypsinogen

15Arg

16Ile

H3N+

Trypsin

chymotrypsin (ACTIVE)

-chimotrypsin(ACTIVE)

Chymotrypsin

Formation of substrate binding pocket

Proteolytic activation of ChymotrypsinProteolytic activation of Chymotrypsin

35

Chymotrypsin: Acid-base catalysisThe catalytic triad

Chymotrypsin: Acid-base catalysisThe catalytic triad

57His

NHN

102 Asp195 Ser

HO

A proton shuttle in the active center

36

Chymotrypsin: Covalent catalysisChymotrypsin: Covalent catalysis

Peptide bond of the food protein

to be cut

O-

C

NH

O C

NH

1st tetrahedral transition intermedier

O

C H2N

1st product

Acyl-enzyme

intermedier+

Ser - OH

His

Ser - O

HisH+

Ser - O

His

37

+ H2O

O-

C OH 2nd tetrahedral transition intermedier

+O

C OH

2nd product

Ser - O

HisH+

Ser - OH

His

38

Specificity of Ser proteasesSpecificity of Ser proteases

O C

NH

CHR

Substrate binding pocket

Chymotrypsin: Big, nonpolar pocketR: aromatic rings (Phe, Tyr, Trp)

Trypsin: negatively charged pocketR: positive charges (Arg,Lys)

Elastase: small pocketR: small (Gly,Ala, Ser)

39

Thrombin is a Serine protease

Proteolytic cleveage (Xa)A

B

S

S

prothrombin

OH

thrombin

fibrinogen

fibrin

40

Enzyme KineticsEnzyme Kinetics

41

S Pv1

v -1

Conditions for measuring initial rate:

[P] 0v1 >>> v -11.

2. [S] 0

Enzyme activity (v) should be measured as initial rate:Enzyme activity (v) should be measured as initial rate:

42

S P[S] decreases[P] increases

S P

equilibrium

[P]

time

Initial rate

S Plinearity

v = constant v decreases v = 0

Product formation as the function of the timeProduct formation as the function of the time

43

The Michaelis-Menten modelThe Michaelis-Menten model

Assumptions:

E + S

k1

k - 1

[ES] E + Pk2

k -2 0IRREVERSIBLE

steadystate

[S] [ES]

One-substrate reaction

Initial rate

44

E + S

k1

k - 1

[ES] E + Pk2

Rate of [ES] formation= Rate of [ES] breakdown

steadystate

Definition: (k2+k-1)/k1= KM

v =vmax [S]

KM + [S]

45

v

[S]

vmax

vmax

12

KM

Vmax = max EA

KM = [S]where v = 1/2 vmax

I. Low [S] II. [S] = KM III. High [S]

Enzyme activity (v, initial rates) as the function of substrate concentration

Enzyme activity (v, initial rates) as the function of substrate concentration

46

v =vmax [S]

KM + [S]v =

vmax

KM

[S]

First order reactionI. Low [S]

II. [S] = KM

Michaelis-Menten Kinetics

III. High [S]

v =vmax [S]

KM + [S]

v = vmax

saturated substrate concentration

Zero order reaction

47

Turnover number kcat =Vmax

[E]

kcat

KM =k-1 + k2

k1

Affinity of the substrate to the enzymelow KM - high affinity

KM Michaelis constant

kcat

KM„catalytic efficiency”

48

Linearization for determination of KM and vmaxLinearization for determination of KM and vmax

1/v

1/[S]

- 1/KM

1/vmax

Lineweaver-Burkdouble reciprocal plot

Slope : Km/Vmax

49

[S] 200 20 2 0.2 0.15 0.013

v 60 60 60 48 45 12

9.1.2.1. Calculate the KM and the vmax from the data below!

v =vmax [S]

KM + [S]

Vmax = 60

12 = 60*0.013

KM + 0.013

Vmax/2=30

KM

50

[S] 200 20 2 0.2 0.15 0.013

v 60 60 60 48 45 12

9.1.2.1. Calculate the Vmax and KM using a graph!

Calculate the reciprocal values!

1/[S] 1/200 and so on

1/v 1/60 and so on

1/v

1/[S]

- 1/KM

1/vmax

1/v

1/[S]

1/v

1/[S]

- 1/KM- 1/KM

1/vmax1/vmax

1/v

1/[S]

- 1/KM

1/vmax

1/v

1/[S]

1/v

1/[S]

- 1/KM- 1/KM

1/vmax1/vmax

51

v= 60*0.1

0.05 + 0.1

Calculate the rate (v) at 0.1 mM and at 100 mM of S,if KM= 0.05 mM, vmax= 60 U

0.1 mM

100 mM v= 60*100

0.05 + 100

v =vmax [S]

KM + [S]v =

vmax [S]

KM + [S]

= 6

0.15= 40 U

= 6000

100.05= 60 U =vmax

52

v= 37*0.2* KM

KM + 0.2* KM

9.1.2.3. Calculate the rate (v) at [S] = 0.2 KM, if the Vmax is 37 U

v =vmax [S]

KM + [S]v =

vmax [S]

KM + [S]

= 37*0.2

1.2= 6.2 U

53

At which [S] will the enzyme show one-quarter of its maximal rate, if its KM=5 mM

v =vmax [S]

KM + [S]v =

vmax [S]

KM + [S]

Vmax/4=Vmax [S]

KM + [S]

(KM + [S]) =4 [S]

Here: [S] = 0.005/3

[S] = KM/(4-1)

54

Classification of enzymes

Classification of enzymes

1. Oxidoreductases

2. Transferases

3. Lyases

4. Isomerases

5. Ligases

55

Nomenclature based on the coenzymes

Nomenclature based on the coenzymes

ATP: kinase

e.g. glucose + ATP glucose-6-P + ADP

glucokinase (hexokinase)

NAD+/NADP+/FAD: dehydrogenase

e.g. lactate + NAD+ pyruvate + NADH + H+

lactate dehydrogenase

56

Phosphatase

Glucose -6P Glucose + Pi

Glucose-6-phosphatase

Phosphorylase

glycogen Glucose -1P+ Pi

Glycogen phosphorylase

57

ISOMERASE / MUTASE / EPIMERASE

Glucose -6Paldohexose

Fructose-6Pketohexose

Phosphohexose isomerase

3-P-glycerate 2-P-glyceratephosphoglycerate mutase

Mannose-5P Glucose-5P2- epimerase

Isomerase: interconversion of an aldo and a ketosugar

Mutase: alters the position of a group

Epimerase: rotates the assimetrical OH group

58

Enzyme Kinetics

Reversible Inhibitions

Enzyme Kinetics

Reversible Inhibitions

59

v

[S]

Competitive InhibitionCompetitive Inhibition

E

E

E

E + S ES E + P

E + I EI

Inhibitor: a substrate analogue binds to the same place

Substrate excess: no inhibition

vmax

12

KM KI

vmax

vmax: No change

KM: increases

60

CH3-CH2-OH CH3-CO

Halcohol

Alcohol dehydrogenase

Etylene glycol

CH2-OH

CH2-OH

CH2-CO

H

CH2-OHoxalate

Methanol/ethylene glycol poisoning:

CH3-OH H- CO

Hmethanol POISON

i.v. alcohol

61

E

E

E

[ES] E + P

[EI]

Noncompetitive InhibitionNoncompetitive Inhibition

[ESI]

E

v

[S]

vmax

12

KM

vmax

vmax: decreases

KM: No change

62

Linearization for determination of KM and vmaxLinearization for determination of KM and vmax

1/v

1/[S]

- 1/KM

1/vmax

Lineweaver-Burkdouble reciprocal plot

Comp. Inh.

Noncomp. Inh.

63

Un-competitive inhibitionUn-competitive inhibition

vmax: decreases

KM: decreases

The inhibitor binds only to the ES complex

[ES]

[ESI]

E

1/v

1/[S]

E E E + P

64

A competitive inhibitor increases/decreases/ does not change the KM of the enzyme

A noncompetitive inhibitor increases/decreases/ does not change the vmax of the enzyme

In case of un-competitive inhibition the inhibitorcan/cannot bind to the enzyme-substrate complex

65

Irreversible inhibitors

Poisons

Irreversible inhibitors

Poisons

66

Irreversible inhibitors of the enzymesIrreversible inhibitors of the enzymes

Specific reaction with the active center (covalent bond)-SH enzymes: monoiodoacetate-OH enzymes: DIPF (see Ser proteases)

Nonspecific inhibitors - denaturation of enzymes - Heavy metal ions- Heat

67

Regulation of enzyme activity

Allosteric regulation


Allosteric regulation

68

E

Apparent competitive inhibitionApparent competitive inhibition

E

E

E + S ES E + P

E + I EI

Inhibitor: NOT a substrate analogueALLLOSTERIC binding siteconformational change

Kinetics: competitive inhibition

69

Allosteric regulationAllosteric regulation

1. Substrate binding site2. Allosteric effector binding site

activator inhibitor

Changes in the conformation

Allosteric enzymes:

70

Conformation change of the enzyme

Decreases(apparent competitive inhibition)

increases

K-type enzymes KM

increases decreases

V-type enzymes Vmax

activation inactivation

71

4

v

[S]

v

[S]

Which of the following graphs shows the kinetics of an allosteric inhibition?

v

[S]

v

[S]

1 2

3

No effectoreffector

72

Allosteric regulationFeed back inhibition of the first specific (irreversible) step

of a pathway

Allosteric regulationFeed back inhibition of the first specific (irreversible) step

of a pathway

A

B C

D

Regulatory role of product “D”

INHIBITION

ACTIVATION

E

F

73

Medication of Hypercholesterolemia

Medication of Hypercholesterolemia

Statins(Mevastatin, Lovastatin)

Competitive analogues of the mevalonate

HMGCoA

cholesterol

REG. step

feed back

mevalonate

Cholesterol metabolism

Reversible inhibitorsMedical applications

Reversible inhibitorsMedical applications

74

Chemotherapy, Antitumor agentsChemotherapy, Antitumor agents

Nucleoside analogues: competitive inhibitors

AIDS - dideoxi nucleosidesColon cancer - 5-fluorouracilleukemia - arabinosyl cytosinesee: selected structures

Folic acid (vitamin) dihydrofolate tetrahydrofolate METOTREXATES solid tumors

Deoxynucleotide synthesis - Dihydrofolate reductase

DNA polymerase, substrates: nucleotides

75

Regulation of enzyme activityEnzyme induction

Regulation of enzyme activityEnzyme induction

cellHormone

nucleusDNA

geneenzyme

Hormone responsive

element

Transcription factor

New synthesis of proteins

76

Regulation of enzyme activityby enzyme induction/repression

Regulation of enzyme activityby enzyme induction/repression

Enzyme (protein)

Amino acids

Protein synthesis breakdown

Inducers: stimulate enzyme synthesisRepressors: enzyme synthesis is inhibited

Protein turnover

77

Regulation of enzyme activityby compartmentation

Regulation of enzyme activityby compartmentation

MitochondrionNADH production

oxidation of NADH: electrontransport chain

CytosolNADH

Lactate production

78

Regulation of enzyme activityby irreversible covalent modification

Regulation of enzyme activityby irreversible covalent modification

Proteolysis: Irreversible activation of proteases

E.g. Serine proteasespancreatic enzymesblood coagulation factors

79

9.1.2.5. [S] mM 0.05 0.067 0.1 0.13 0.27 0.53mU, no inhib. 0.40 0.50 0.60 0.66 0.80 0.88mU, with inhib. 0.17 0.20 0.24 0.27 0.32 0.36

0.60 = Vmax*0.1

KM + 0.1

0.50 = Vmax*0.067

KM + 0.067

no inhib.

0.24 = Vmax*0.1

KM + 0.1

0.20 = Vmax*0.067

KM + 0.067

with inhib.

Calculations, 9.1.2.5.

80REG

catalyticsubunits

REG

REG

Regulation of enzyme activity: Allosteric regulation - Cooperativity

Regulation of enzyme activity: Allosteric regulation - Cooperativity

81

Homotrop cooperativityHomotrop cooperativity

The catalitic subunit binds the substrate

this will activate the other catalitic subunits

substrate = activatorsubunits are equal

TT formhigh KM

+S

low KM

RR form

+S

RR form

T: less active( „tense” form)

R: more active (relaxed form)

82

Kooperativity: sigmoid graph

Kooperativity: sigmoid graph

v

[S]The enzyme works in a narrow concentration range

KM increases

Aspartate transcarbamoylase:Homotrop cooperativity

Aspartate transcarbamoylase:Homotrop cooperativity

Interaction of 6 catalytic subunits:s s s

T forms

S S S

R forms

83

Heterotrop cooperativityHeterotrop cooperativity

The regulatory subunit binds the allosteric effector

activates/inactivates the catalitic subunits

substrate = activatorsubunits are different

More active

+I

I

I

Less active

84

Aspartate transcarbamoylase: 6 regulatory subunitsPositive (ATP) and negative (CTP) heterotrop effect

Aspartate transcarbamoylase: 6 regulatory subunitsPositive (ATP) and negative (CTP) heterotrop effect

Sigmoid graph (cooperativity)

v

[S]

ATP+CTP-

Activator(ATP): less sigmoid

Inhibitor(CTP): more sigmoid graph

lower KM

higher KM

-

Feed back inhibition

Pathway

CTP

Asp + carbamoyl-P

ATP +

85

PathwayGlc

PFK

Dual role of ATP: Substrate AND inhibitore.g. phosphofructokinase (PFK)

Dual role of ATP: Substrate AND inhibitore.g. phosphofructokinase (PFK)

ATP (energy)

-

Feed back inhibition of the product

+ATP

v

[ATP]

86

• The same reaction is catalyzed

• Differences– Distribution among organs– Different genes– quantitative parameters (v; KM)– substrate specificity– regulation

IsoenzymesIsoenzymes

87

Isoenzymes: e.g. glucokinase/hexokinase

Isoenzymes: e.g. glucokinase/hexokinase

v

[glucose]KMKM

hexokinase

glucokinase

KM = 0.5 mM

KM = 10 mM

high affinity

low affinity

88

Different KM - different phase of working activity

GUT Hyperglycaemiaafter meal

liver

glucokinase

RBChexokinase

Glc utilizationat normal/low level

Glc utilizationonly at high level

89

At which [S] will glucokinase show one-quarter of its maximal rate, if its KM=10 mM

v =vmax [S]

KM + [S]v =

vmax [S]

KM + [S]

Vmax/4=Vmax [S]

KM + [S]

[S] = 3.3 mM

1

4=

[S]

10 + [S]

90

Isoenzymes: Lactate dehydrogenaseIsoenzymes: Lactate dehydrogenase

CO O–

C

CH3

O

CO O–

C

CH3

HHO

NADH + H+

NAD+

pyruvate lactate

4 subunits (two types: H and M)

5 izoenzymes: H4 H3M H2M2 HM3 M4

Characteristic tissue distribution: H4: heartM4: liver, HxMx: muscle

Nonfunctional plasma enzymestissue damage: appearance in the serum diagnosis

91

Regulation of enzyme activity: Hormonal regulation

Regulation of enzyme activity: Hormonal regulation

Covalent modification:

Enzyme phosphorylation / dephosphorylation

CHANGES IN THE CONFORMATION

Activation / inactivation

92

Mechanism of enzyme phosphorylationMechanism of enzyme phosphorylation

+ ATP E

P

+ ADP

Dephosphorylated enzyme Phosphorylated enzyme

E

Ser - O - P OO -

O -

Covalent bond (ester bond)

Protein kinase A (PK A)(cAMP dependent)

E

Phosphorylation site

93

cAMP is an ancient signal of hungercAMP is an ancient signal of hunger

Blood

glucagon(hormon)

hypo-glycaemia(starvation)

LIVER CELL

ATP

cAMP

AC: adenylate cyclase

AC

E

P

HR

R: hormon receptor

Starvation:all enzymes phosphorylated(activated or inactivated)

PK A

E

94

Phosphorylated enzymes can be either activated or inactivated

Phosphorylated enzymes can be either activated or inactivated

Blood

low glc

glucagon

LIVER produces glc from glycogen

glycogen

P inactive

P active

95

Well fed state: insulinWell fed state: insulin

Blood

hyper-glycaemia

insulin(hormon)

LIVER CELL

E

P

HR

R: hormon receptor

Well fed:all enzymes dephosphorylated(activated or inactivated)

protein phosphatase

PiE

96

Blood

high glc

insulin

LIVER produces glycogen from glc

glycogen

active

inactive

97


Complex regulation(allosteric + hormonal)


Complex regulation(allosteric + hormonal)

98

Phosphorylase kinasePactive

EpinephrineHormonal regulation

STRESS Muscle contraction

Ca2+


ATP glycogen

EnergyP active

Muscle: glycogen breakdown in exerciseMuscle: glycogen breakdown in exercise

99

: catalitic subunit

partially active

-PP-

FULLY ACTIVE

-PP-

Ca2+

Ca2+

epinephrine

inactive

Ca2+PK A

: regulatory subunits, phosphorylation site

Ca2+

glycogen

EnergyP active

Phosphorylase kinase

Muscle contraction

Ca2+


STRESS

Pactive

Complex regulation of phosphorylase kinaseComplex regulation of phosphorylase kinase

(calmoduline): regulatory subunit, Ca2+ binding site

100

glycogen

EnergyP active


Muscle contraction

Ca2+


STRESS

Pactive


ATP Complex regulation of Glycogen phosphorylaseComplex regulation of Glycogen phosphorylase

TT

ATP glucose

RRPP

TTPP


101

glycogen

EnergyP active


Muscle contraction

Ca2+


STRESS

Pactive


ATP

PFK I

ATP

PFK I : Dual role of ATPPFK I : Dual role of ATP

E

Phosphofructokinase

active center

Fructose-6-P

ATP

Allosteric site

EATP

Higher KM

102

Regulation of Enzyme Activity Summary

Regulation of Enzyme Activity Summary

1. Allosteric regulation - intracellular - fastmain effectors: ATP/ADP, NAD+/NADH, end products

2. Hormonal regulation - slower Enzyme phosphorylation/dephosphorylation

activation or inactivation

3. Enzyme induction - new enzyme synthesis - slowestinduction of gene expression (hormonal effect)

4. Irreversible activation by proteolysis

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ENZYMES