7 Enzyme

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ENZYMES - Basics

Substrate Product

Aim: You should get a solid understanding of basic

concepts of enzyme catalysis and enzyme kinetics

Enzymes are EFFICIENT

Enzyme Reaction Uncatalysed

(kuncat s-1)

Catalysed

(kcat s-1)

Rate enhancement

(kcat/kuncatat)

OMP-decarboxylase Decarboxylation 2.8 x10-16 39 1,4 x 1017

-9 11,

Carboanhydrase CO2-hydratisation 1,3 x10-1 1 x106 7,7 x 106

Orotidine

monophosphate

Uridine

monophosphate

Orotidine monophosphate decarboxylase

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Enzymes are SPECIFIC

-

SPECIFICITY varies:

Trypsin: R1 = Lys/Arg

Thrombin: R1 = Arg, R2 = Gly

Very HIGH specificity: DNA-polymerase I adds the correct nucleotide with an

,

Cofactor - Coenzyme - Cosubstrate Prosthetic group

Many enzymes use ions/small molecules

during catalysis: COFACTORS

2 KINDS OF

COFACTORS

METAL IONS e.g. Zn2+

COENZYMES Small org. molecules, e.g. NAD+

The cofactor can be tightly bound to the enzyme PROSTHETIC GROUP

The coenzyme can be free like a substrate and is then also called

COSUBSTRATE. Example NAD+.

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Six enzyme classes

EC-number (Enzyme Commission)

Example: Chymotrypsin = EC 3.4.21.1

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Enzymes catalyse by facilitating the formation of

transition state

Uncatalysed

ubstrate roduct

Enzyme catalysed

S+E ES ES* P + E

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Active site Catalysis always includes the formation of

ENZYME-SUBSTRATE COMPLEX

The substrate binds to anACTIVE SITE

X-Ray

Evidence: Fluorescence

Saturation kinetics

General properties of an Active site

Active site is a cleft

Walls from different regions

bstrat

The substrate is bound with weak bonds

Specificity is based upon the exact fit

Binding by lock and key

orinduced fit

Su

The active site can supply an environmentfree of water

Catalytic groups in the active site:

- side chain in the protein part

- cofactor

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Enzyme kinetics

E + S ES E + Pk1

k-1

k2

k-2Simple model:

Michaelis-Menten kineticsE + S ES E + P

k1k-1

k2

You can formulate the Michelis-Menten equation

assuming STEADY-STATE kinetics

V= Vmax[S]

KM + [S]

V = the initial reaction rate

Vmax = the maximal rate = k2 [E]tot

KM = the Michaelis-Menten constant KM =k1 +

k2

k1

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Michaelis-Menten kinetics

E + S ES E + Pk1

k-1

k2

V= Vmax[S]

KM + S

What do Vmax

and kcat

mean?

E + S ES E + Pk1

k-1

k2

Vmax = k2[E]tot

Vmax = kcat[E]tot

k2 or kcat is called the turnover number

Says what the enzyme can do at maximal conditions

Enzyme Function kcat

Carboanhydrase Hydratises CO2 600 000 s-1

Lactate dehydrogenase Oxidises lactate 1 000 s-1

Lysozyme Open up cell walls 0,5 s-1

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What is the meaning of KM?

E + S ES E + P

k1

k-1

k2

KM : substrate concentration

at V = 1/2 Vmax

KM

=1+

2

k1

Low KM

indicates that the

enzyme has high affinity for the

substrate

What is the meaning of kcat/KM?

The cell often works at substrate concentrations BELOW KM. kcat is

not good as indicator of efficiency under those circumstances

kcat/KM is a good indicator of efficiency in the cell

V= Vmax

[S]

[S] +KM

= kcat

[E]tot

[S]

[S]+KM

=

kcat

KM

[S][E]tot

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How do you determine KM och Vmax?

V= Vmax[S] 1

V=

1

Vmax

+KM

Vmax

1

[S]

Lineweaver-Burk

Determine V

for a number

of [S]

Enzyme inhibition

A. Competitive inhibition

EI-complex

Competitive inhibition can beCOUNTERACTED with more

substrate)][

1(][

max

i

M

K

IKS

VV

++

=

Ki = Kdiss for the EI-complex

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Enzyme inhibition

B. Non-competitive inhibition

-

Non-competitive inhibition cant

be diminished by more substrate

][

)

][

1(

max

SK

K

IV

M

i

+

+

=

Ki = Kdiss for the EI-complex

- -ONAGONAMONAGONAM - -

H O

Lysozyme hydrolyses bacterial cell walls

- -ONAGONAMOH + HONAGONAM- -

O

CH2OH

H

OH HH

HO

CH2OH

H

H

O HH

O

OH

OH

: -ace y muram c ac

CCH3

H

COO

N H

CO

CH3

HH N H

CO

CH3-

NAG: N-acetylglucosamine

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The active site of Lysozyme

Site D distorts the sugar

A B C D E F

LysozymeCleaved bond

E H OH

E

The catalytic mechanism of Lysozyme

Glu

35COOH

D

Asp

52-OOC

+ H2O

35-

D

Asp

52-OOC

+

Glu

35COOH Asp

52-OOC

Important items:General acid catalysis

D

OH H Carbo-cation stabilisedelectrostatically by Asp 52

Formation of carbo-cation

favoured by the steric effect

in site D

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Enzyme activity is controlled

Ver im ortant for the cell to be

E. coli

able to control the enzyme

activity!

Different ways to contro l enzyme activ ity

1. synthesise (transcription/translation)

A. Make new enzyme molecules degrade (= slow control)

. syn es se a par cu ar soenzyme

3. degrade the enzyme

1.Al losteric contro l

- often feed-back inhibition

B. Control of the activity of existing enzymes (= fast control)

. - e.g. phosphorylation/dephosphorylering of the

enzymmolekylen

3. Proteolytic activation

- part of the polypeptide is removed

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Phosphorylation is made by kinases

Dephosphorylation by phosphatases

+ ATPProtein kinase

+ ADP

Part of

Ser, Thr

or Tyr

Protein

phosphatase

Phosphorylation AND dephosphorylation are both strongly

pushed to the right!

Proteolytic cleavage activates proteins

Some enzymes/proteins are synthesised as inactive

pro-enzymes = zymogens.

They are cleaved and refolded to active proteins not until

they are needed

The cleavage can occur outside the cell

Example:

H drol tic enz mes

Blood coagulation

Hormones, e.g. Proinsulin becomes insulin

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Enzymes in washing powder

Why?

- lower temperature

- energy is saved

Which enzymes?

-

- Lipases degrade fats

- Amylases degrade carbohydrates

- Cellulases degrade cellulose

Treatment of cotton fibres to facilitatedyeing

In order to dye cotton fibres non-cellulose material

(pectin) must be removed

It is done with alkaline solutions

The enzyme pectin lyase can be used instead

ea s o an env ronmen a y e er me o Requires lower temperature (saves energy)

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Summary

Enzymes are efficient and specific

Cofactor - Coenz me - Cosubstrate - Prosthetic rou

6 enzyme classes - EC-number

Enzymes perform catalysis by facilitating the formation

of the transition state

Active site

Michaelis-Menten kinetics

KM, Vmax, kcat and kcat/KM Enzyme inhibition

Enzyme mechanism

Control of enzymatic activity

Technical use of enzymes

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7 Enzyme