Azin Nowrouzi, PhD

Tehran University of Medical Sciences

TUMS

Chemical reactionChemical reactionA BCatalyst

Reactant(s) Product(s)

What are some of the known catalysts?

HeatAcid BaseMetals

Catalysts•Increase the rate of a reaction.•Are not consumed by the reaction.•Can act repeatedly.

A +B B + CCatalyst

Enzyme is either a pure protein or Enzyme is either a pure protein or may require a non-protein portionmay require a non-protein portion

• Apoenzyme = protein portion• Apoenzyme + non-protein part = Holoenzyme

According to Holum, the non-protein portion may be: • A coenzyme - a non-protein organic substance which is

loosely attached to the protein part.• A prosthetic group - an organic substance which is firmly

attached to the protein or apoenzyme portion.• A cofactor - these include K+, Fe++, Fe+++, Cu++, Co++, Zn+

+, Mn++, Mg++, Ca++, and Mo+++.

Basic enzyme reactionsBasic enzyme reactions

S + E E + PS = Substrate P = Product E = Enzyme

Swedish chemist Savante Arrhenius in 1888 proposed:

Substrate and enzyme form some intermediate substance known as The Enzyme-Substrate Complex (ES):

S + E ESES P + E

Binding step

Catalytic step

There are two models of enzyme substrate interactionThere are two models of enzyme substrate interaction1. Lock and key model Emil Fischer (1890)

2. Induced fit model Daniel Koshland (1958)

The active site:• Substrate Binding Site• Catalytic Site

Induced fit in Carboxypeptidase AInduced fit in Carboxypeptidase A

Three amino acids are located near the active site (Arg 145, Tyr 248, and Glu 270)

Enzyme-Substrate complex is transientEnzyme-Substrate complex is transient

When the enzyme unites with the substrate, in most cases the forces that hold the enzyme and substrate are non-covalent.Binding forces of substrate are:

• Ionic interactions: (+)•••••(-)• Hydrophobic interactions: (h)•••••(h)• H-bonds: O-H ••••• O, N-H ••••• O, etc. • van der Waals interactions

ESS + E P + E

Some important characteristics of enzymesSome important characteristics of enzymes1. Potent (high catalytic power) High reaction rates

– They increase the rate of reaction by a factor of 103-1012

2. Efficient (high efficiency)– catalytic efficiency is represented by Turnover number.

moles of substrate converted to product per second per mole of the active site of the enzyme

3. Milder reaction conditions Enzymatically catalyzed reactions occur at mild temperature, pressure, and nearly neutral pH. (i.e physiological conditions)

4. Specific (specificity)– Substrate specific– Reaction Specific– Stereospecific

5. Capacity for regulationEnzymes can be activated or inhibited so that the rate of product formation responds to the needs of the cell.

• Location within the cellMany enzymes are located in specific organelles within the cell. Such compartmentization serves • to isolate the reaction substrate from competing reactions, • to provide a favorable environment for the reaction, and • to organize the thousands of enzymes present in the cell into purposeful

pathqways.

SpecificitySpecificity• Substrate Specificity

1. Absolute specificity: For example Urease

2. Functional Group specificity: For example OH, CHO, NH2.

3. Linkage specificity: For example Peptide bond.

• Reaction specificity– Yields are nearly 100%– Lack of production of by-products– Save energy and prevents waste of metabolites

• Stereospecificity – Enzymes can distinguish between enantiomers and

isomers

Enzymes requiring metal ions as cofactorsEnzymes requiring metal ions as cofactors

Many vitamins are coenzyme precursorsMany vitamins are coenzyme precursors

Methods for naming enzymes Methods for naming enzymes (nomenclature)(nomenclature)

1. Very old method: Pepsin, Renin, Trypsin

2. Old method: Protease, Lipase, Urease

3. Systematic naming (EC = Enzyme Commission number ):

The name has two parts:

The first part: name of substrate (s)

The second part: ending in –ase, indicates the type of

reaction.

Additional information can follow in parentheses: L-malate:NAD+ oxidoreductase (decarboxylating)

Each enzyme has aEach enzyme has a EC number number == Enzymenzyme Commissionommission numbernumber

• EC number consists of 4 integers • The 1st designates to which of the six major classes an

enzyme belongs.• The 2nd integer indicates a sub class, e.g. type of bond• The 3rd number is a subclassification of the bond type or

the group transferred in the reaction or both (a susubclass)

• The 4th number is simply a serial number

Enzyme EC number

Alcohol dehydrogenase 1.1.1.1

Arginase 3.5.3.1

Pepsin 3.4.21.1

There are six functional classes of enzymesThere are six functional classes of enzymes

Class Names Functions

1 Oxidoreductases AH + NAD+ A+ + NADH

2 Transferases A-X + B A + B-X

3 Hydrolases A-OX + H2O A-OH + HOX

4 Lyases R1R2R3CCR4R5R6 R1R2C==CR4R5 + R3 + R6

5 Isomerases trans cis, L-form D-form, etc.

6 Ligases Formation of C-C, C-S, C-O, C-N bonds by condensation reactioncoupled to ATP hydrolysis

EC 3 Hydrolases  Function

EC 3.1 Acting on ester bonds

EC 3.2 Glycosylases

EC 3.3 Acting on ether bonds

EC 3.4Acting on peptide bonds (peptidases)

EC 3.5Acting on carbon-nitrogen bonds, other than peptide bonds

EC 3.6 Acting on acid anhydrides

EC 3.7 Acting on carbon-carbon bonds

EC 3.8 Acting on halide bonds

EC 3.9Acting on phosphorus-nitrogen bonds

EC 3.10 Acting on sulfur-nitrogen bonds

EC 3.11Acting on carbon-phosphorus bonds

EC 3.12 Acting on sulfur-sulfur bonds

EC 3.13 Acting on carbon-sulfur bonds

EC5 Isomerases

 

EC 5.1Racemases and epimerases

EC 5.2 cis-trans-Isomerases

EC 5.3Intramolecular isomerases

EC 5.4Intramolecular transferases (mutases)

EC 5.5 Intramolecular lyases

EC 5.99 Other isomerases

Enzyme Nomenclature and ClassificationEC Classification

Class

Subclass

Sub-subclass

Serial number

Example of Enzyme NomenclatureExample of Enzyme Nomenclature

• Common name(s)– Invertase, sucrase

• Systematic name -D-fructofuranoside fructohydrolase (E.C. 3.2.1.26)

• Recommended name -fructofuranosidase

Kinetic

Energy barrier = Free Energy of ActivationEnergy barrier = Free Energy of Activation

T = Transition state

(Ea)

Thermodynamics:Type (Exergonic or Endergonic)

Kinetics:How fast the reaction will proceed

X T* Y

What’s the difference? Many enzymes function by lowering the activation energy of reactions.

Adapted from Alberts et al (2002) Molecular Biology of the Cell (4e) p.166

Enzyme Stabilizes Transition StateEnzyme Stabilizes Transition State

EEAA = Activation energy ; a barrier to the reaction = Activation energy ; a barrier to the reaction

Can be overcome by adding energy.......

......or by catalysis

Enzymes Are Complementary to Transition StateEnzymes Are Complementary to Transition State

If enzyme just binds substrate then there will be no further reaction

Enzyme not only recognizes substrate, but also induces the formation of transition state

X

Active Site Is a Deep Buried PocketActive Site Is a Deep Buried Pocket

Why energy required to reach transition stateis lower in the active site?

(1) Stabilizes transition(2) Expels water(3) Reactive groups(4) Coenzyme helps

(2)

(3)(4)

(1)CoE

+

-

Juang RH (2004) BCbasics

Active Site Avoids the Influence of WaterActive Site Avoids the Influence of Water

Preventing the influence of water sustains the formation of stable ionic bonds

Adapted from Alberts et al (2002) Molecular Biology of the Cell (4e) p.115

-+

Modes of rate enhancementModes of rate enhancement

• Facilitation of Proximity– Increase the Effective concentration.– Hold reactants near each other in proper

orientation

• Strain, Molecular Distortion, and Shape Change– Put a strain on susceptible bonds

• General Acid –Base Catalysis– Transfer of a proton in the transition state

• Covalent Catalysis– Form covalent bond with substrate destabilization

of the substrate.

Factors affecting rate of enzyme reactionsFactors affecting rate of enzyme reactions

1. Temperature

2. pH

3. Enzyme concentration [E]

4. Substrate concentration [S]

5. Inhibition

6. Regulation (Effectors)

• Little activity at low temperature (low number of collisions)• Rate increases with temperature (more successful collisions)

– rate doubles for every 10°C increase in temperature• Most active at optimum temperatures (usually 37 C in humans) • Enzymes isolated from thermophilic organisms display maxima

around 100°C • Enzymes isolated from psychrophilic organisms display maxima

around 10°C.• Activity lost with denaturation at high temperatures

1- Optimum Temperature1- Optimum Temperature

2- Optimum 2- Optimum pHpH• Effect of pH on ionization of active site.• Effect of pH on enzyme denaturation.• Each enzyme has an optimal pH (~ 6 - 8 )

– Exceptions :

digestive enzymes in the stomach( pH 2) digestive enzymes intestine (pH 8)

3- Enzyme concentration3- Enzyme concentration• The Rate (v) of reaction Increases proportional to

the enzyme concentration [E] ([S] is high).

• When enzyme concentration is constant, increasing [S] increases the rate of reaction, BUT

• Maximum activity reaches when all E combines with S (when all the enzyme is in the ES, ,form).

4- Substrate concentration4- Substrate concentration

21 3 4 5 6 7 80

0 2 4 6 8

Substrate (mole) [S]

Pro

duct

(v)

80

60

40

20

0

S+E

(in a fixed period of time)

Constant [E]

EnzymeVelocityCurve

P

Juang RH (2004) BCbasics

Michaelis-Menten equationMichaelis-Menten equation

S Ek1

k-1

ESk2 P

0

1

2

3

4

5

0 10 20 30 40 50

v, µ

mo

l/m

in

[S], mM

0.5Vmax

Km

maximal velocity, Vmax

MM equation derivation (steady state)MM equation derivation (steady state)

Practical SummaryPractical Summary- V- Vmaxmax and K and Kmm

Enzyme Substrate Km (mM)

Catalase H2O2 1,100

Chymotrypsin Gly-Tyr-Gly 108

Carbonic anhydrase CO2 12

Beta-galactosidase D-lactose 4

Acetylcholinesterase acetylcholine (ACh) 0.09

• Vmax– How fast the reaction can occur under ideal circumstances.

• Km – Range of [S] at which a reaction will occur. – Binding affinity of enzyme for substrate

• LARGER Km the WEAKER the binding affinity

• Kcat / Km – Practical idea of the catalytic efficiency, i.e. how often a

molecule of substrate that is bound reacts to give product.

1. When [S] << Km

vo = (Vmax / Km )[S]

2. When [S] = Km

vo = Vmax /2

3. When [S] >> Km

vo = Vmax

Order of reactionOrder of reaction

2

zero order

First order

Mixed order

Importance of VImportance of Vii

in measurement of Enzyme activityin measurement of Enzyme activity

Working with vo minimizes complications with1. reverse reactions2.product Inhibition

The rate of the reaction catalyzed by an enzymein a sample is expressed in Units. Units = V = activity = Micromoles (mol; 10-6 mol or ….),of [S] reacting or [P] produced/min.

It is better to measure it at linear part of the curve

S E ESk2

Pk1

k-1

Lineweaver-Burk plotLineweaver-Burk plot

1

vKm

Vmax

1

[S]

1

Vmax

Km S

vo

1/S

1vo

Double reciprocal Direct plot

1Vmax

- 1 Km

1/2

Jua

ng

RH

(2

00

4)

BC

ba

sicsv

Vmax [S]

Km [S]

Allosteric enzymesAllosteric enzymes• Why the sigmoid shape? • Allosteric enzymes are multi-subunit enzymes,

each with an active site. • They show a cooperative response to substrates

Sigmoidal curve

hyperbolic curve michaelis-menten

kinetics

Irreversible Inhibition = Enzyme Irreversible Inhibition = Enzyme stops working permanentlystops working permanently

1. Destruction of enzyme2. Irreversible Inhibitor = forms covalent bonds to E

(inactive E) Examples:

– Diisopropylfluorophosphate• inhibits acetylcholine esterase • binds irreversibly to –OH of serine residue

– Cyanide and sulfide • Inhibit cytochrome oxidase • bind to the iron atom

– Fluorouracil• inhibits thymidine synthase (suicide inhibition - metabolic

product is toxic )– Aspirin

• Inhibits prostaglandin synthase • acylates an amino group of the cyclooxygenase

Reversible Inhibition = Temporary decrease of enzyme function

• Three types based on “how increasing [S] affects degree of inhibition”:

1. Competitive – degree of inhibition decreases

2. Non-competitive – degree of inhibition is unaffected

3. Anti- or Uncompetitive – degree of inhibition increases

The Lineweaver-Burk plot is useful in determining the mechanisms of actions of various inhibitors.

The Effects of Enzyme InhibitorsThe Effects of Enzyme Inhibitors

ExampleExample• When a slice of apple is exposed to air, it quickly

turns brown. This is because the enzyme o-diphenyl oxidase catalyzes the oxidation of phenols in the apple to dark-colored products.

• Catechol can be used as the substrate The enzyme converts it into o-quinone (A), which is then further oxidized to dark products.

ExperimentsExperiments

  Tube A Tube B Tube C Tube D

[S] 4.8 mM 1.2 mM 0.6 mM 0.3 mM

1/[S] 0.21 0.83 1.67 3.33

Δ OD540

(Vi)0.081 0.048 0.035 0.020

1/Vi 12.3 20.8 31.7 50.0

 Tube

ATube

BTube

CTube

D

[S]4.8 mM

1.2 mM

0.6 mM

0.3 mM

1/[S] 0.21 0.83 1.67 3.33

ΔOD

540

(Vi)

0.060

0.032

0.019

0.011

1/Vi 16.7 31.3 52.6 90.9

 Tube A Tube B Tube C Tube D

[S] 4.8 mM 1.2 mM 0.6 mM 0.3 mM

1/[S] 0.21 0.83 1.67 3.33

ΔOD540

(Vi)0.040 0.024 0.016 0.010

1/Vi 25 41 62 102Effect of phenylthiourea

Effect of para-hydroxybenzoic acid (PHBA)

No Inhibitor

0

0.5

1

1.5

2

2.5

-0.6 -0.4 -0.2 0 0.2 0.4 0.6 0.8 1

1/v

, /µ

mo

l/min

1/[S], /mM

-1/Km

1/Vmax

Km/Vmax

0

1

2

3

4

5

0 10 20 30 40 50

v, µ

mo

l/min

[S], mM

0.5Vmax

Km

I- Competitive InhibitionI- Competitive Inhibition

Competitive S ECI S + E ES E + P

I+

EI

Kic

0

1

2

3

4

5

0 10 20 30 40 50

v, µ

mo

l/min

[S], mM

No I

+ C I

Km

0.5Vmax

Kmapp

1

vKm

Vmax

1

[S]

1

Vmax

v Vmax [S]

Km [S]v

Vmax [S]

Km [S]

1

v

Km

Vmax

1

[S]

1

Vmax

1[I]

Kic

0

0.5

1

1.5

2

2.5

-0.6 -0.4 -0.2 0 0.2 0.4 0.6 0.8 1

1/v

, /µ

mo

l/min

1/[S], /mM

+ C I

No I

-1/Km

1/Vmax

Km/Vmax

-1/Kmapp

Kmapp/Vmax

0

0.5

1

1.5

2

2.5

-0.6 -0.4 -0.2 0 0.2 0.4 0.6 0.8 11

/v, /

µm

ol/m

in1/[S], /mM

-1/Km1/Vmax

Km/Vmax

II- Noncompetitive InhibitionII- Noncompetitive Inhibition

Noncompetitive(mixed-type)

0

1

2

3

4

5

0 10 20 30 40 50

v, µ

mo

l/min

[S], mM

0.5Vmax

Km

S ENCI

NCI +

Kic Kiu

S + E ES E + P

I

EI ESI

I+S E

v Vmax [S]

Km [S]

1

vKm

Vmax

1

[S]

1

Vmax

1

v

Km

Vmax

1

[S]

'

Vmax

v Vmax [S]

Km ' [S]

1[I]

Kic

' 1[I]

Kiu

0

1

2

3

4

5

0 10 20 30 40 50

v, µ

mo

l/min

[S], mM

No I

+ NCI

Km

0.5Vmax

0

0.5

1

1.5

2

2.5

-0.6 -0.4 -0.2 0 0.2 0.4 0.6 0.8 11

/v, /

µm

ol/m

in1/[S], /mM

+ NC I

No I-1/Km

1/Vmax

Km/Vmax

Km/Vmaxapp

1/Vmaxapp

0.5Vmax

0

0.5

1

1.5

2

2.5

-0.6 -0.4 -0.2 0 0.2 0.4 0.6 0.8 1

1/v

, /µ

mo

l/min

1/[S], /mM

-1/Km1/Vmax

Km/Vmax

III- Uncompetitive InhibitionIII- Uncompetitive Inhibition

Uncompetitive(catalytic) S E UCI

Kiu

S + E ES E + P

ESI

I+

v Vmax [S]

Km [S]1

vKm

Vmax

1

[S]

1

Vmax

v Vmax [S]

Km ' [S]1

vKm

Vmax

1

[S]

'

Vmax

' 1[I]

Kiu

0

1

2

3

4

5

0 10 20 30 40 50

v, µ

mo

l/min

[S], mM

0.5Vmax

Km

0

1

2

3

4

5

0 10 20 30 40 50

v, µ

mo

l/min

[S]. mM

No I

+ UC I

Km

0.5Vmax

Kmapp

0.5Vmax

0

0.5

1

1.5

2

2.5

-0.6 -0.4 -0.2 0 0.2 0.4 0.6 0.8 1

1/v

, /µ

mo

l/min

1/[S]. /mM

+ UC I

No I

-1/Km

1/Vmax

Km/Vmax

1/Vmaxapp

-1/Kmapp

Kmapp/Vmax

app

Enzyme inhibitors in medicineEnzyme inhibitors in medicine

• Many current pharmaceuticals are enzyme inhibitors (e.g. HIV protease inhibitors for treatment of AIDS)

• An example: Ethanol is used as a competitive inhibitor to treat methanol poisoning.

Methanol formaldehyde (very toxic)

Ethanol competes for the same enzyme.

Administration of ethanol occupies the enzyme thereby delaying methanol metabolism long enough for clearance through the kidneys.

Alcohol dehydrogenase

Aminotransferases

Aspartate aminotransferase

(AST or SGOT)

Alanine aminotransferase

(ALT, or SGPT)

Myocardial infarction

Viral hepatitis

Lactate Dehydrogenase (LDH) myocardial infarction

Creatine Kinase (CK) Myocardial infarc., brain,

skeletal muscle disorder

Cholinesterase Liver, erythrocytes

Gamma-glutamyltransferase Liver damage

Acid phosphatase Carcinoma of prostate

Alkaline phosphatase (AP) Bone disease

Lipase Acute pancreatitis

Ceruloplasmin Hepatolenticular degeneration (wilson’s disease)

Alpha-amylase Intestinal obstruction Som

e di

agno

stic

ally

impo

rtan

t enz

ymes

Useful enzymes for early diagnosis of dental caries and

periodontal disease

Isozymes of lactate dehydrogenaseIsozymes:

– Are catalitically identical (have same catalytic activity) BUT physically distinct

– Can be detected by gel electrophoresis (different electrical charge)– Occur in oligomeric enzymes like lactate dehydrogenase (LDH)

In LDH• Protomers H and M can combine to make five different

tetramers.

Isoenzymes of Creatine kinase• CK has 3 forms dimer

B and M chains: • CK1= BB• CK2= MB• CK3=MM• Heart only tissue rich in

CK2, increases 4-8 hr after chest pains- peaks at 24 hr.

• LDH peaks 2-3 days after MI.

• New markers: Troponin T, Troponin I

5- Regulation (Effectors)5- Regulation (Effectors)Effectors can be classified:

According to type:• Homotropic effector: Substrate itself is the effector

• Heterotropic effector: substance other than substrate is the effector

According to their effect:• Activators (positive effectors)

– Increase the rate of enzyme

• Inhibitors (negative effectors)– Decrease the velocity of reaction – Stop the enzyme

IrreversibleReversible

CompetitiveNon-competitiveUncompetitive

Increase or decrease in enzyme reaction rate is reflected in the graph of V versus S

Metabolic pathways

• A metabolic pathway is a chain of enzymatic reactions– Most pathways have many steps, each having a

different enzyme (E1, E2, E3, E4)– Step by step, the initial substance used as substrate

by the first enzyme is transformed into a product that will be the substrate for the next reaction

• Metabolic regulation is necessary to: – maintain cell components at appropriate levels.– conserve materials and energy.

Regulation of “Enzyme activity”A. Regulation at trascription level (slowest)

B. Isozymes: Regulation specific to distinct tissues and developmental stages

C. Compartmentation of S, E and P

D. Specific Proteolytic Cleavage

E. Covalent Modification (Reversible phosphorylation or adenylation)

F. In response to metabolic products (fastest)1. Substrate level control

2. Product Inhibition

3. Feedback control

4. Allosteric Effectors

A. Regulation at transcription level

1. Regulation of [E] by • Gene repression• Induction of genetic expression of enzyme

2. There is competition in a cell between the processes of protein synthesis and protein destruction.

• By altering these rates, one can alter the whole cell catalytic rate.

3. It is rather slow

B. Isoenzymes

• Isozymes Provide a Means of Regulation Specific to Distinct Tissues and Developmental Stages

• Differential expression of isozymes

• LDH (for example)

• Preferential substrate affinity

C. Compartmentalization of enzymes

Substrates and cofactors within the cell are also compartmentalized

Examples:• Enzymes of glycolysis are located in the cytoplasm• Enzymes of citric acid cycle are in the mitochondria• Hydrolytic enzymes are found in the lysosome

but the release of these suicide enzymes during apoptosis is an on/off switch than a true regulation.

D. Proteolytic activation

Activation of a zymogen. • Some enzymes are secreted as inactive

precursors, called zymogens. • Pancreatic proteases - trypsin, chymotrypsin,

elastase, carboxypeptidase are all synthesized as zymogens - trypsinogen, chymotrypsinogen, proelastase and procarboypeptidase

• Clotting factors are also part of a proteolytic cascade

• Hormone peptides (Proinsulin Insulin)• an on/off switch more than regulation.

E. Covalent modification

Reversible phosphorylation

Phosphorylation is the most common type of modification

Two important classes of enzymes are:– Kinases Add a phosphate group to

another protein/enzyme (phosphorylation) • transfer of phosphoryl group from ATP to -OH

group of serine, threonine or tyrosine

– Phosphatases Remove a phosphate group from a protein/enzyme (dephosphorylation)

1- Control of [S]• Concentration of substrate and product

also control the rate of reaction, providing a biofeedback mechanism.

• Usually,

0.1Km<[SPhysiology]<10km

Change in enzyme activity

Mild changes in [S]

Homotropic effectors – substrate itself (binding at different site than the active site) affects enzyme activity on other substrate molecules. Most often this is a positive effector.

2- Product inhibition• Enzyme is reversibly inhibited by the product.

Example: hexokinase - first reaction in glycolysis, hexokinase is inhibited by glucose-6-phosphate (G6P, the product)

glucose + ATP glucose-6-phosphate + ADP 

_

Why?As v approaches Vmax, the product becomes significant, and can compete with the substrate for the enzyme The product becomes a competitive inhibitor and slows down activity of the enzyme.

A B C D E

3- Negative Feedback control(End product inhibition)

• Final product of a metabolic sequence feeds-back negatively on early steps

• In feedback inhibition, there is a second binding site on the enzyme where the inhibitor binds, so that the inhibitor is not necessarily similar in structure to the substrate.

What happens?• As the need for product E decreases, E will accumulate • Most efficient to inhibit at first step of the pathway, slow the first

reaction so intermediates do not build up  • An increase in the concentration of E, leads to a decrease in its rate

of production of E.

Enz 2Enz 1 Enz 3 Enz 4

_

                                                     

  

1. Simple feed-back inhibition. The final product (E) inhibits the step from A to B.

2. Co-operative feed-back inhibition. Both final products (D, E) inhibit the first step of their own synthesis together.

3. Multivalent feed-back inhibition.

                                                 

4. Inhibition at a ramification of a biosynthesis pathway (sequential inhibition)

Regulation of the metabolism, feed-back inhibition by the final product - end product inhibition

4- Positive Feedforward control

• Earlier reactants in a metabolic sequence feed-forward positively on later steps.

Metabolism involves the complex integration of many feedback and feedforward loops.

+

+If A is accumulating, it speeds up downstream reactions to use it up.

4- Allosteric control • Allosteric activator stabilizes active "R" state

– shift the graph to the left • Allosteric inhibitor stabilizes less active or inactive "T" state

– shift the graph to the right

Multi reactant enzymes reactancy

• Published by W. W. Cleland in1963

• Nomenclature is based on number of substrates and products in the reaction.

• Reactancy: the number of kinetically significant substrates or products and designated by syllables Uni, Bi, Ter, Quad.

A P Uni Uni

A P + Q Uni Bi

A + B P + Q Bi Bi

A + B + C P + Q + R + S Ter Quad

Multi reactant enzymes mechanism

Sequential - if all S add to E before any P are released.– Sequential ordered - if S add in an obligatory

order (two on; two off).– Sequential random - if S do not add in

obligatory order (two on; two off).

Ping Pong - If one or more S released before all S bind• (one on, one off; one on, one off); • Note: there is some sort of modified enzyme

intermediate (often covalent intermediate).

Random sequential (example)

Ordered sequential (example)

Ping pong or double displacement mechanism

Double displacement (example)

Other enzymes

• Some ribonucleoprotein enzymes have been discovered.– The catalytic activity is in the RNA part.– They are called Ribozymes

• Catalytic antibodies are called Abzymes.

• In competitive inhibition the inhibitor is similar in structure to the substrate and binds to the enzyme at the active site, preventing the substrate from binding. In feedback inhibition, the inhibitor binds to the enzyme at a site away from the active site and acts by altering the shape of the enzyme in such a way that it is incapable of catalyzing the reaction. Feedback inhibition is a natural part of the process by which an organism regulates the chemical reactions that take place in its cells. In that sense it is done on purpose. Competitive inhibition usually involves inhibitors, commonly called poisons, that do not belong in the cell.

•  

Enzymes may be regulated by

1..

2.Competitive product inhibition and allosteric regulation (fastest). • Many enzymes are inhibited by either their

products, or by other chemicals, often those from further down a metabolic pathway.

• Such enzymes may be 'gatekeepers' to a specific branch of metabolism,

Biochemical reaction

A BCatalyst

Reactant Product

What are the biocatalysts ( Enzymes)? Proteins & RNA ???

When the chemical reaction occurs in a biological system it is called a biochemical reaction.

Biological system: Mild conditionsSimultaneous presence of different substances Specific needs in specific times

ES ES Pk1

k-1

k2

Basic enzyme reaction

A B

S P

S + E ES (Enzyme-substrate complex)

ES P + E

Catalyst

Enzyme

Substrate Product

Reactant Product

Binding step Catalytic step