Lecture 8-Properties Classification & Kinetics.ppt

(1)

Enzymes

Properties, Classification

(2)

Definition

Enzyme

Biological catalyst (normally a protein,

rarely RNA) that functions to speed up

the rate of a biological reaction, but is

not altered or consumed in the reaction.

(3)

Consider A Hypothetical Reaction

 Reaction spontaneously proceeds due to random kinetic Reaction spontaneously proceeds due to random kinetic

energy of reactants

energy of reactants ((vibrational, rotational, vibrational, rotational, translational

translational).).

 Reactants eventually collide with sufficient force Reactants eventually collide with sufficient force

(activation energy)

(activation energy) to undergo a reaction. to undergo a reaction.

 Heat increases the amount of kinetic energy and rate of Heat increases the amount of kinetic energy and rate of

reaction.

 Enzymes eliminate some of the Enzymes eliminate some of the ““randomnessrandomness”” of of

ATP

+

H

₂₂

O



(Reactants)

(Reactants) (Products)_(Products)

ADP

OHOH

P

i

i HH

+

(4)

Introductory Terminology

 Substrates Substrates – – the substances upon which an enzyme acts the substances upon which an enzyme acts

(i.e. reactants).

 ProductsProducts – the substances produced by chemical – the substances produced by chemical

modification of substrates.

 Active SiteActive Site – the specific region on/in an enzyme where – the specific region on/in an enzyme where

substrates bind and where the catalytic reaction occurs.

 Transition StateTransition State – the unstable ( – the unstable (““energizedenergized””) )

intermediate formed in an enzymic reaction that has

properties of both the substrate and the product.

 Also, the point in a reaction where reactants & products have Also, the point in a reaction where reactants & products have

the highest energy.

Activation Energy

(5)

Example of the Transition State

(Alcohol Dehydrogenase)

HC – C – OH

HC – C – OHH HH H

H H

H H HC – C – OHC – C – O

H H

H H HC – C = OHC – C = O

H H

H

Transition State

Transition State

(Properties of both substrate and product) (Properties of both substrate and product)

Ethanol

Ethanol AcetaldehydeAcetaldehyde

(6)

-Enzymes lower activation energy by . . .

Enzymes lower activation energy by . . .



Orienting & holding substrates very close

together.



Creating & stabilizing the transition state

intermediate.



Facilitating the reaction via reactive amino

acids in the active site.

(7)

Models for Enzyme Action

The

“

Lock & Key

”

Model

 Proposed by Emil Fischer (1894).Proposed by Emil Fischer (1894).

 The enzyme active site (i.e. lock) perfectly matches the The enzyme active site (i.e. lock) perfectly matches the

shape of the substrate (i.e. key).

 The enzyme thus allows only one substrate to bind to The enzyme thus allows only one substrate to bind to

(key) (key)

(8)

Models for Enzyme Action

The

“

Induced Fit

”

Model

The Induced fit

The Induced fit

binding of glucose

by hexokinase.

 Proposed by Daniel Koshland (1958).Proposed by Daniel Koshland (1958).

 Subtrates fit into active site like a flexible Subtrates fit into active site like a flexible ““hand-in-glovehand-in-glove””..  Enzyme-Substrate binding (interactions) changes the shape Enzyme-Substrate binding (interactions) changes the shape

of both enzyme & substrate to fit snugly.

(9)

Energy Changes

During the Progress of a Reaction

 Products and reactants have Products and reactants have

different amounts of energy.

different amounts of energy.  Reactants pass through a Reactants pass through a

transition state as they form

products.

 Enzymes lower the activation Enzymes lower the activation

energy (

energy (GG‡_{) but not the free}_{) but not the free}

energy (

energy (GG).).

 ““Activation EnergyActivation Energy”” vs. vs. ““Free Free

Energy

Energy””

 _{Significance of}_{Significance of}_G_G_º_º_{= G}_{= G}

2

2 – G – G11??

(G

(G₁₁))

(G

(10)

Six Classes of Enzymes

1.

1. OxidoreductasesOxidoreductases – catalyze oxidation-reduction – catalyze oxidation-reduction

reactions; add/remove electrons (& protons) from its

substrate.

 Includes dehydrogenases, oxidases, reductases, peroxidases etc.Includes dehydrogenases, oxidases, reductases, peroxidases etc.  Example:Example: Alcohol dehydrogenase. Alcohol dehydrogenase.

CH

CH₃₃CHCH₂₂- OH- OH CHCH₃₃CHCH O

O

II

NAD

NAD++ NADHNADH ++ HH++

Ethanol

Ethanol

Acetaldehyde

(11)

Polyphenol Oxidase

Another Example of an Oxidoreductase

(Flavonoids)

(Tyrosine)

(3,4-Dihydroxy-PHE)

Melanins

Brown Pigmentation in Plants & Animals

Polyphenol Oxidase

(12)

Six Classes of Enzymes

2.

2. TransferasesTransferases – transfer a functional group from one – transfer a functional group from one

molecule to another (amino, phosphoryl, methyl, acyl).

molecule to another (amino, phosphoryl, methyl, acyl).  Includes transaminases, transmethylases, acyl transferases, etc.Includes transaminases, transmethylases, acyl transferases, etc.  Example:Example: Hexokinase. Hexokinase.

(

(AA-P-P-P-P-P-P)) ((AA-P-P-P-P))

Hexokinase

(13)

Six Classes of Enzymes

3.

3. HydrolasesHydrolases – cleave bonds by adding a water molecule. – cleave bonds by adding a water molecule.

 Includes esterases, phosphatases, peptidases, lipases, Includes esterases, phosphatases, peptidases, lipases,

glycosidases.

 Example:Example: Phospholipase A Phospholipase A

2

2..

H H C C I I C C I I C C H H H H Choline

Choline -- Pi_Pi -

-- O -- C -- (CH

- O - C - (CH₂₂))_n_nCHCH₃₃ O

O

II

- O - C - (CH

- O - C - (CH₂₂))_n_nCHCH₃₃

II

O

H

HHH

H H C C I I C C I I H H H H Pi -

-- O -- C -- (CH

- O - C - (CH₂₂))_n_nCHCH₃₃ O O II II - OH - OH

HO - C - (CH

HO - C - (CH₂₂))_n_nCHCH₃₃ O O II II + + (Phosphatidylcholine) (Phosphatidylcholine)

H - OH

(Fatty Acid)

(14)

Six Classes of Enzymes

4.

4. LyasesLyases – remove functional groups via non-hydrolytic – remove functional groups via non-hydrolytic

reactions.

 Often result in formation of a double bond. Often result in formation of a double bond.

 Includes decarboxylases, deaminases, dehydratases.Includes decarboxylases, deaminases, dehydratases.  Example:Example: Phenylalanine Ammonia Lyase. Phenylalanine Ammonia Lyase.

NH

NH₂₂

COOH

NH

NH₃₃

COOH

(15)

Six Classes of Enzymes

4.

4. LyasesLyases – remove functional groups via non-hydrolytic – remove functional groups via non-hydrolytic

reactions.

 Often result in formation of a double bond. Often result in formation of a double bond.

 Includes decarboxylases, deaminases, dehydratases.Includes decarboxylases, deaminases, dehydratases.  Example: Example: Isocitrate Lyase.Isocitrate Lyase.

COO I CH₂

I

C COO I CH I COO -H -HO COO I CH₂ I

C COO H -H -CH I COO O= COO I CH₂ I

C COO I CH I COO -H -HO COO I CH₂ I

C COO H -H -COO I CH₂ I

(16)

5.

5. IsomerasesIsomerases – catalyze rearrangements of – catalyze rearrangements of

functional groups within a molecule.

 MutasesMutases – transfer functional groups from one – transfer functional groups from one

position to another.

 EpimerasesEpimerases – invert functional groups about – invert functional groups about

asymmetric carbons.

 ExampleExample – Triose Phosphate Isomerase. – Triose Phosphate Isomerase.

CH

CH22OHOH I I C=O C=O I I CH

CH₂₂- PO- PO₄₄

CH

I

C - OH

I

CH

(17)

5.

5. IsomerasesIsomerases – catalyze rearrangements of – catalyze rearrangements of

functional groups within a molecule.

 MutasesMutases – transfer functional groups from one – transfer functional groups from one

position to another.

 EpimerasesEpimerases – invert functional groups about – invert functional groups about

asymmetric carbons.

(18)

Six Classes of Enzymes

6.

6. LigasesLigases – use the energy from ATP hydrolysis to form – use the energy from ATP hydrolysis to form

bonds between two substrate molecules.

bonds between two substrate molecules.  Form C-C, C-S, C-O & PO_{Form C-C, C-S, C-O & PO}

3

32-2- ester bonds. ester bonds.

 Includes synthetases, carboxylases.Includes synthetases, carboxylases.  Example:Example: Acetyl-CoA Carboxylase. Acetyl-CoA Carboxylase.

CH

CH3 3 - C - CoA- C - CoA O

O

II

O

CO

CO₂₂

Acetyl-CoA

ATP

(19)

Enzyme Commission Nomenclature

(EC Numbers)

Every enzyme has a unique number series that precisely

Every enzyme has a unique number series that precisely

describes its chemical reaction.

EC 1, 2, 3, 4, 5 & 6 = oxidoreductases, transferases,

hydrolases, lyases, isomerases & ligases, respectively.

 EC 3EC 3 – enzymes are – enzymes are hydrolaseshydrolases (use water to break up a (use water to break up a

molecule) molecule)

 EC 3.4EC 3.4 – are hydrolases that act on – are hydrolases that act on peptide bondspeptide bonds..  EC 3.4.11EC 3.4.11 – are those hydrolases that cleave off the – are those hydrolases that cleave off the N-

N-terminal amino acid

terminal amino acid from a polypeptide. from a polypeptide.

(20)

Enzymes Often Require Cofactors

Cofactor

– loosely bound non-protein components

of enzymes that assist in catalytic reactions.

Two main Types:

 Organic:Organic: Also call Also call ““co-enzymesco-enzymes”” (e.g. CoA, NAD, (e.g. CoA, NAD,

Biotin).

 Inorganic:Inorganic: Assorted mineral ions (e.g. Mg Assorted mineral ions (e.g. Mg2+2+, Zn, Zn2+2+).).

Note:

Note: ““Prosthetic groupsProsthetic groups”” are tightly bound non-protein are tightly bound non-protein (usually organic) components of proteins. (e.g. heme).

(21)

Enzyme Kinetics

The Quantitative Study

of Enzyme Catalysis

(22)

How do they get that

liquidy syrup inside that candy?

 Fructose is more soluble & sweeter than sucrose.Fructose is more soluble & sweeter than sucrose. Sucrose

Sucrose

+ water

= Paste

Glucose + fructose

+ water

= Syrup

= Syrup Invertase

(23)

Some Industrial Enzymes

 Cellulase: Cellulase: Hydrolyzes cellulose; used as digestive aid, & Hydrolyzes cellulose; used as digestive aid, &

in biofuel production.

 Collagenase:Collagenase: Hydrolyzes collagen; promotes burn and Hydrolyzes collagen; promotes burn and

wound healing.

 Invertase:Invertase: Hydrolyzes sucrose; used in the manufacture Hydrolyzes sucrose; used in the manufacture

of soft-centered candy.

 Lipase:Lipase: Hydrolyzes lipids; used as a digestive aid; Hydrolyzes lipids; used as a digestive aid;

improves flavor of cheese.

(24)

Objectives of Enzyme Kinetics

To understand . . .



Velocities (rates) of enzymic reactions.



Strength of substrate binding (affinity).



Mode of regulation of enzyme activity.



Effects of inhibitors.



Equilibrium point.

(25)

Substrate Concentration

Has the Greatest Effect

(No Substrate, No Reaction)

V

V_max_max = the maximum possible = the maximum possible velocity that a reaction can

velocity that a reaction can

have (at infinite substrate

concentration).

½ V

½ V_max_max = the half-maximal = the half-maximal velocity of a reaction.

velocity of a reaction.

K

K_m_m = the substrate = the substrate

concentration at which the

enzyme has half-maximal

velocity.

Velocity Vmax

V_max 2

(26)

Initial Velocity is Fastest

 Substrate concentrations Substrate concentrations

gradually decrease (i.e.

they are consumed in the

reaction).

 Reaction velocities slow Reaction velocities slow

down with time.

 Reverse reaction starts to Reverse reaction starts to

occur.

 Initial velocities (V_{Initial velocities (V}

0

0) are ) are

most relevant.

V

el

o

ci

(27)

Mathematical Units for Velocity

Concentration Based



mmoles,



moles, nmoles



substrate consumed, product produced



per sec, min, hr.



per unit of protein

(mg)



(per reaction volume)

e.g. Lactate e.g. Lactate Dehydrogenase Dehydrogenase



molesmoles

min

(28)

Importance of V

_max_max

& K

_m_m



Both are constants that help define enzymes.



Enzymes rarely operate at true V

max

.

 [S] in most cells not high enough.[S] in most cells not high enough.

 Little or no regulation by [S] at VLittle or no regulation by [S] at V

max max..

 [S] at K[S] at K

m

m is more realistic. is more realistic.



K

m

(Michaelis Constant) is an indication of

enzyme affinity for substrate.

(29)

Significance of K

m

 Enzymes with lower KEnzymes with lower K m

m’’s have greater substrate affinity.s have greater substrate affinity.

V

Vmaxmax

V

V_max_max

2

Decreasing

(30)

Significance of K

m

 Enzymes with lower KEnzymes with lower K m

m’’s have greater substrate affinity.s have greater substrate affinity.

P

ATP

+

P

P PP

Glucose-6P Glucose

Fructose-6P Fructose-1,6P₂

Hexokinase

P-Fructokinase

K_{m ATP} = 0.15 – 1.0

(31)

Experimental Determination

of

V

_max_max

& K

_m_m

Substrate

Substrate

Concentration

V

el

o

ci

ty

V

el

o

ci

(32)

The Michaelis-Menton Equation

A Mathematical Expression for a Hyperbolic Plot

[

S

]

Velocity

Vmax Vmax

2

K_m

V_max [S] [S] + K_m

(33)

Lineweaver-Burk Plots

Linear Transformations of Michaelis-Menton Plots



Often referred to as

“

double reciprocal

”

plots.



Derived by taking the reciprocal of both sides of

the Michaelis-Menton equation.

V

Vmaxmax [[SS]]

[

[SS] + K] + K_m_m

V =

(34)

Derivation of

the Lineweaver-Burk Equation

V

Vmaxmax [[SS]]

[

[SS] + K] + K_m_m

V =

V

V_max_max [[SS]] [

[SS] + K] + K_m_m

1 1 == V V + + 1 1

== [[SS]] KKmm

+ + 1 1 == V

V VV_max_max 1

1 ₁₁

[

[SS]] V

V_max_max

K

K_m_m

x x + + 1 1 == V

V VV_max_max

1

[

[SS]] V

V_max_max

K

K_m_m

x

y = mx + b

X

(35)

Derivation of

the Lineweaver-Burk Equation

[S] V_max

V_max 2

K_m

V

(36)

Additional Kinetics Terminology

Enzyme Unit (I.U.):

Enzyme Unit (I.U.): the amount of enzyme that produces the amount of enzyme that produces 1 1



molemole of product per minute. of product per minute.

 KatalKatal (SI unit) = amount of enzyme that produces 1 mole of (SI unit) = amount of enzyme that produces 1 mole of

product per second.

Specific Activity:

Specific Activity: moles of product formed per minute, moles of product formed per minute, per mg protein (i.e.

per mg protein (i.e. mol / minmol / minmg).mg).

Turnover Number (K

Turnover Number (K_cat_cat; Catalytic Constant): ; Catalytic Constant): the number the number of substrate molecules converted to product per unit time

of substrate molecules converted to product per unit time

by a single molecule of enzyme

by a single molecule of enzyme ( (under saturating under saturating substrate conc

substrate conc.). .). Mathematically, KMathematically, K_cat_cat = V = V_max_max/[Enz]./[Enz].

Catalytic Efficiency (K

(37)

Kinetic Parameters of Selected Enzymes*

Enzyme

Enzyme No. (KNo. (KTurnover Turnover _cat_cat) s) s-1-1 KK_m_m((M)M)

Catalytic Eff.

(K

(K_cat_cat/K/K_m_m) M) M-1-1_s_s-1-1

Catalase Catalase

2 H

2 H₂₂OO₂₂ 2 H 2 H₂₂OO₂₂+ O+ O₂₂ 40,000,00040,000,000 1,100,0001,100,000 1.5 x 101.5 x 1077

Carbonic Anhydrase Carbonic Anhydrase

CO

CO₂₂  H H₂₂COCO₃₃ 1,000,0001,000,000 26,00026,000 4.0 x 104.0 x 1077

Acetylcholinesterase Acetylcholinesterase

Acetylcholine

Acetylcholine  Acetate + Choline Acetate + Choline 14,00014,000 0.90.9 16.0 x 1016.0 x 1077

Triose-Pi Isomerase Triose-Pi Isomerase

Gald-3P

Gald-3P  DHAP DHAP 4,3004,300 0.180.18 24.0 x 1024.0 x 1077

Fumarase Fumarase

Fumarate

Fumarate  Malate Malate 800800 5.05.0 16.0 x 1016.0 x 1077

x

(38)

Sample Questions

1.

1. Use the Michaelis-Menton equation to determine the Use the Michaelis-Menton equation to determine the

speed (i.e. velocity) of a reaction where the enzyme V

speed (i.e. velocity) of a reaction where the enzyme V_max_max

= 10 mols/min, [S] = 2 mM, & Km = 3 mM.

2.

2. Use a Lineweaver-Burk double reciprocal plot to Use a Lineweaver-Burk double reciprocal plot to

determine the theoretical V

determine the theoretical V_max_max and K and K_m_m of an enzymatic of an enzymatic reaction.

(39)

Study Questions / Objectives

1.

1. Describe the general characteristics of enzymes and enzymic Describe the general characteristics of enzymes and enzymic

reactions.

2.

2. Compare & contrast the Compare & contrast the ““Lock & KeyLock & Key”” vs. vs. ““Induced FitInduced Fit”” models for models for

enzyme action.

3.

3. Name, describe & exemplify the six classes of enzymes.Name, describe & exemplify the six classes of enzymes.

4.

4. Describe & exemplify the various types of enzyme cofactors.Describe & exemplify the various types of enzyme cofactors.

5.

5. Describe some of the main objectives of enzyme kinetics studies.Describe some of the main objectives of enzyme kinetics studies.

6.

6. What is the meaning and significance of VWhat is the meaning and significance of V_max_max & K & K_m_m..

7.

7. Distinguish between the Michaelis-Menton and Lineweaver-Burke Distinguish between the Michaelis-Menton and Lineweaver-Burke

equations and plots, and their applications.

8.

8. Distinguish between the following terms: enzyme unit, specific Distinguish between the following terms: enzyme unit, specific

activity, turnover number, catalytic efficiency.