enzymes • chemical kinetics and catalysis, • kinetics of the … · 2018. 3. 22. · catalysis...

Enzymes Chemical kinetics and catalysis; Kinetics of the simple

enzymatic reactions; Chemical aspects of enzyme action

Mitesh Shrestha

Catalysis

A catalyst is a substance that changes the rate of a reaction by lowing the activation energy, Ea. It participates a reaction in forming an intermediate, but is regenerated.

Enzymes are marvelously selective catalysts.

A catalyzed reaction, NO (catalyst) 2 SO2 (g) + O2 — 2 SO3 (g)

via the mechanism i 2 NO + O2 2 NO2 (3rd order) ii NO2 + SO2 SO3 + NO

Uncatalyzed rxn

Catalyzed rxn

rxn

Energy

Homogenous vs. heterogeneous catalysts

A catalyst in the same phase (gases and solutions) as the reactants is a homogeneous catalyst. It effective, but recovery is difficult.

When the catalyst is in a different phase than reactants (and products), the process involve heterogeneous catalysis. Chemisorption, absorption, and adsorption cause reactions to take place via different pathways.

Platinum is often used to catalyze hydrogenation

Catalytic converters reduce CO and NO emission.

Enzymes

• Macromolecular Biological Catalyst

• Proteins that increase the rate of reaction by

lowering the energy of activation

• They catalyze nearly all the chemical reactions taking place in the cells of the body.

• Not altered or consumed during reaction.

• Reusable

Enzymes Are Classified into six functional Classes (EC number Classification) by the International Union of

Biochemists (I.U.B.). on the Basis of the Types of

Reactions That They Catalyze

• EC 1. Oxidoreductases

• EC 2. Transferases

• EC 3. Hydrolases

• EC 4. Lyases

• EC 5. Isomerases

• EC 6. Ligases

Principle of the international classification

Each enzyme has classification number

consisting of four digits:

Example, EC: (2.7.1.1) HEXOKINASE

• EC: (2.7.1.1) these components indicate the following groups of enzymes:

• 2. IS CLASS (TRANSFERASE)

• 7. IS SUBCLASS (TRANSFER OF PHOSPHATE)

• 1. IS SUB-SUB CLASS (ALCOHOL IS PHOSPHATE ACCEPTOR)

• 1. SPECIFIC NAME

ATP,D-HEXOSE-6-PHOSPHOTRANSFERASE (Hexokinase)

H O

OH

H

OHH

OH

CH2OH

H

OH

H H O

OH

H

OHH

OH

CH2OPO32

H

OH

H

23

4

5

6

1 1

6

5

4

3 2

ATP ADP

Mg2+

glucose glucose-6-phosphate

Hexokinase

1. Hexokinase catalyzes:

Glucose + ATP glucose-6-P + ADP

Oxidoreductases, Transferases and Hydrolases

Lyases, Isomerases and Ligases

EC 1. Oxidoreductases

• Biochemical Activity: –Catalyse Oxidation/Reduction Reactions Act

on many chemical groupings to add or remove hydrogen atoms.

• Examples: – Lactate dehydrogenase. –Glucose Oxidase. –Peroxidase. –Catalase. –Phenylalanine hydroxylase.

1. Oxidoreductases

• Catalyze oxidation-reduction reactions

- oxidases - peroxidases - dehydrogenases

EC 2. Transferases

• Biochemical Activity: – Transfer a functional groups (e.g. methyl or

phosphate) between donor and acceptor molecules.

• Examples: – Transaminases (ALT & AST). – Phosphotransferases (Kinases). – Transmethylases. – Transpeptidases. – Transacylases.

2. Transferases

• Catalyze group transfer reactions

EC 3. Hydrolases

• Biochemical Activity:

– Catalyse the hydrolysis of various bonds Add water across a bond.

• Examples:

– Protein hydrolyzing enzymes (Peptidases).

– Carbohydrases (Amylase, Maltase, Lactase).

– Lipid hydrolyzing enzymes (Lipase).

– Deaminases.

– Phosphatases.

3. Hydrolases

• Catalyze hydrolysis reactions where water is the acceptor of the transferred group

- esterases - peptidases - glycosidases

EC 4. Lyases

• Biochemical Activity:

–Cleave various bonds by means other than hydrolysis and oxidation.

–Add Water, Ammonia or Carbon dioxide across double bonds, or remove these elements to produce double bonds.

• Examples:

– Fumarase.

–Carbonic anhydrase.

4. Lyases

EC 5. Isomerases

• Biochemical Activity: –Catalyse isomerization changes within a

single molecule. –Carry out many kinds of isomerization:

• L to D isomerizations. • Mutase reactions (Shifts of chemical

groups). • Examples:

– Isomerase. –Mutase.

5. Isomerases

• Catalyze isomerization reactions

EC 6. Ligases

• Biochemical Activity:

– Join two molecules with covalent bonds Catalyse reactions in which two chemical groups are joined (or ligated) with the use of energy from ATP.

• Examples:

–Acetyl~CoA Carboxylase.

–Glutamine synthetase

6. Ligases (synthetases)

• Catalyze ligation, or joining of two substrates

• Require chemical energy (e.g. ATP)

Chemical reactions

• Chemical reactions need an initial input of energy = THE ACTIVATION ENERGY

• During this part of the reaction the molecules are

said to be in a transition state.

Reaction pathway

Making reactions go faster

• Increasing the temperature make molecules move faster

• Biological systems are very sensitive to temperature changes.

• Enzymes can increase the rate of reactions without increasing the temperature.

• They do this by lowering the activation energy.

• They create a new reaction pathway “a short cut”

An enzyme controlled pathway

• Enzyme controlled reactions proceed 108 to 1011 times faster than corresponding non-enzymic reactions.

Enzymes

Lower a

Reaction’s

Activation

Energy

Structure of enzymes

Enzymes

Complex or holoenzymes (protein part and nonprotein part – cofactor)

Simple (only protein)

Apoenzyme (protein part) Cofactor

Prosthetic groups

-usually small inorganic molecule or atom;

-usually tightly bound to apoenzyme

Coenzyme

-large organic molecule

-loosely bound to apoenzyme

Enzyme structure

• Enzymes are proteins

• They have a globular shape

• A complex 3-D structure

Human pancreatic amylase

The active site

• One part of an enzyme, the active site, is particularly important

• The shape and the chemical environment inside the active site permits a chemical reaction to proceed more easily

Cofactors

• An additional non-protein molecule that is needed by some enzymes to help the reaction

• Tightly bound cofactors are called prosthetic groups

• Cofactors that are bound and released easily are called coenzymes

• Many vitamins are coenzymes Nitrogenase enzyme with Fe, Mo and ADP cofactors

Jmol from a RCSB PDB file © 2007 Steve Cook H.SCHINDELIN, C.KISKER, J.L.SCHLESSMAN, J.B.HOWARD, D.C.REES

STRUCTURE OF ADP X ALF4(-)-STABILIZED NITROGENASE COMPLEX AND ITS

IMPLICATIONS FOR SIGNAL TRANSDUCTION; NATURE 387:370 (1997)

The substrate

• The substrate of an enzyme are the reactants that are activated by the enzyme

• Enzymes are specific to their substrates

• The specificity is determined by the active site

The Lock and Key Hypothesis

• Fit between the substrate and the active site of the enzyme is exact

• Like a key fits into a lock very precisely

• The key is analogous to the enzyme and the substrate analogous to the lock.

• Temporary structure called the enzyme-substrate complex formed

• Products have a different shape from the substrate

• Once formed, they are released from the active site

• Leaving it free to become attached to another substrate

The Lock and Key Hypothesis

Enzyme may

be used again Enzyme-

substrate

complex

E

S

P

E

E

P

Reaction coordinate

The Lock and Key Hypothesis

• This explains enzyme specificity

• This explains the loss of activity when enzymes denature

The Induced Fit Hypothesis

• Some proteins can change their shape (conformation)

• When a substrate combines with an enzyme, it induces a change in the enzyme’s conformation

• The active site is then moulded into a precise conformation

• Making the chemical environment suitable for the reaction

• The bonds of the substrate are stretched to make the reaction easier (lowers activation energy)

The Induced Fit Hypothesis

• This explains the enzymes that can react with a range of substrates of similar types

Hexokinase (a) without (b) with glucose substrate http://www.biochem.arizona.edu/classes/bioc462/462a/NOTES/ENZYMES/enzyme_mechanism.html

Factors affecting Enzymes

• substrate concentration

• pH

• temperature

• inhibitors

Substrate concentration: Non-enzymic reactions

• The increase in velocity is proportional to the

substrate concentration

Reaction

velocity

Substrate concentration

Substrate concentration: Enzymic reactions

• Faster reaction but it reaches a saturation point when all the enzyme molecules are occupied.

• If you alter the concentration of the enzyme then Vmax will change too.

Reaction

velocity

Substrate concentration

Vmax

The effect of pH

Optimum pH values

Enzyme

activity Trypsin

Pepsin

pH

1 3 5 7 9 11

The effect of pH

• Extreme pH levels will produce denaturation

• The structure of the enzyme is changed

• The active site is distorted and the substrate molecules will no longer fit in it

• At pH values slightly different from the enzyme’s optimum value, small changes in the charges of the enzyme and it’s substrate molecules will occur

• This change in ionisation will affect the binding of the substrate with the active site.

The effect of temperature

• Q10 (the temperature coefficient) = the increase in reaction rate with a 10°C rise in temperature.

• For chemical reactions the Q10 = 2 to 3 (the rate of the reaction doubles or triples with every 10°C rise in temperature)

• Enzyme-controlled reactions follow this rule as they are chemical reactions

• BUT at high temperatures proteins denature • The optimum temperature for an enzyme controlled

reaction will be a balance between the Q10 and denaturation.

The effect of temperature

Temperature / °C

Enzyme

activity

0 10 20 30 40 50

Q10 Denaturation

The effect of temperature

• For most enzymes the optimum temperature is about 30°C

• Many are a lot lower, cold water fish will die at 30°C because their enzymes denature

• A few bacteria have enzymes that can withstand very high temperatures up to 100°C

• Most enzymes however are fully denatured at 70°C

Inhibitors

• Inhibitors are chemicals that reduce the rate of enzymic reactions.

• The are usually specific and they work at low concentrations.

• They block the enzyme but they do not usually destroy it.

• Many drugs and poisons are inhibitors of enzymes in the nervous system.

The effect of enzyme inhibition

• Irreversible inhibitors: Combine with the functional groups of the amino acids in the active site, irreversibly.

Examples: nerve gases and pesticides, containing organophosphorus, combine with serine residues in the enzyme acetylcholine esterase.

The effect of enzyme inhibition

• Reversible inhibitors: These can be washed out of the solution of enzyme by dialysis.

There are two categories.

The effect of enzyme inhibition

1. Competitive: These compete with the substrate molecules for the active site.

The inhibitor’s action is proportional to its concentration.

Resembles the substrate’s structure closely.

Enzyme inhibitor

complex Reversible

reaction

E + I EI

The effect of enzyme inhibition

Succinate

Fumarate + 2H++ 2e- Succinate dehydrogenase

CH2COOH

CH2COOH CHCOOH

CHCOOH

COOH

COOH

CH2

Malonate

The effect of enzyme inhibition

2. Non-competitive: These are not influenced by the concentration of the substrate. It inhibits by binding irreversibly to the enzyme but not at the active site.

Examples

• Cyanide combines with the Iron in the enzymes cytochrome oxidase.

• Heavy metals, Ag or Hg, combine with –SH groups.

These can be removed by using a chelating agent such as EDTA.

Michaelis–Menten Curve

Michaelis–Menten equation

The Michaelis–Menten equation describes how the (initial) reaction rate V0 depends on the position of the substrate-binding equilibrium and the rate constant k2.