ENZYMES

Classification Mechanism of enzyme action Coenzymes Specificity of enzymes Isoenzymes Factors affecting rate of enzyme reactions Enzyme inhibitors

Enzyme
Definition
As proteins produced by the cells of living organisms which act as biological catalysts in controlling and accelerating the rate of biochemical reactions in the cell at fairly low temperatures.

Terms and Definition

Apoenzyme : enzyme consists of a protein part Holoenzyme : enzyme consists of a nonprotein
part Coenzyme : organic nonprotein part of a holoenzyme, Prosthetic group : coenzymes attached firmly to apoenzymes Activator : inorganic nonprotein part of a holoenzyme that is loosely attached to a apoenzyme

Enzymes are proteins which control biochemical reactions in cells

All enzymes are globular proteins and round in

shape They have the suffix "-ase" Intracellular enzymes are found inside the cell. Extracellular enzymes act outside the cell (e.g. digestive enzymes)

Activator
Zymogen : enzymes secreted in an
inactive form and requires a specific agent to remove the blocking peptide that covers the active site. Active enzyme can act as activator of its own zymogen. E.g. Pepsin and trypsin. Pepsinogen is activated by H+ ions and by pepsin to form pepsin. This action is called autocatalysis.

Enzymes are catalysts speed up chemical reactions

Reduce activation energy required to start a reaction between molecules Substrates (reactants) are converted into products Reaction may not take place in absence of enzymes (each enzyme has a specific catalytic action) Enzymes catalyse a reaction at max. rate at an optimum state

Characteristics of Enzyme
(1) As biological catalysts
lowering the activation energy, EA and speed up reaction

Characteristics of Enzyme
(2) Specificity
Each enzyme is limited to one specific reaction that involves a specific substrate only because of the specific fit between enzyme and substrate. The small functional area on the enzyme where the substrate or substrates attach to is called the active site.

Characteristics of Enzyme
(3) As protein Enzymes are usually very large globular proteins which are usually bigger than its substrate molecule.

Classification of Enzymes
The nomenclature committee of the International Union of Biochemistry and Molecular Biology (IUBMB) recommended enzymes to be classified under 6 classes:
(1) (2) (3) (4) (5) (6) Oxidoreductases Transferases Hydrolases Lyases Isomerases Ligases

Classification of Enzymes
EC Name of class Catalyses
a variety of oxidation-reduction reactions
transfers of groups (methyl, phosphate,etc.) hydrolysis reactions to split into smaller molecules by the addition of water the cleavage of C-C, C-O, C-S and C-N bonds other than hydrolysis or oxidation.

Example
oxidase, catalase, dehydrogenase
DNA polymerase protease, nuclease

1
2 3

Oxidoreductase
Transferase Hydrolase

Lyase

decarboxylase

5
6

Isomerase
Ligase

atomic rearrangements within a molecule.

the reaction which joins two molecules

epimerase
DNA ligase

How enzymes work?

(1) Reaction mechanism

E + S ES EP E + P
In an enzyme-catalysed reaction, the substrate (S) first binds to the active site of the enzyme (E) to form an enzyme-substrate (ES) complex, then the substrate is converted into product while attached to the enzyme (EP), and finally the product (P) is released while enzyme remained unchanged.

Energy Change in Chemical Reactions

Difference in the energy levels between
products and reactants of a chemical reactions is called the free energy. 3 types of chemical reaction: (1) exergonic or exothermic reaction (2) endergonic or endothermic reaction (3) isoergonic or isothermic reaction

(1) exergonic / exothermic reaction

An exergonic reaction is a chemical reaction where the variation of
Gibbs free energy is negative. This tells us the direction that the reaction will follow. At constant temperature , constant pressure an exergonic reaction is signified by condition:

which describes a chemical reaction that releases energy in the form

of heat, light, etc. Exergonic reactions are a form of exergonic processes in general or spontaneous processes and the opposite is called an endergonic reaction. Exergonic reactions are said to occur spontaneously but this does not imply that the reaction will take place unconstrained. For instance, the reaction between hydrogen and oxygen is very slow and not observed in absence of a suitable catalyst.

(2) endergonic / endothermic reaction

also called an unfavorable reaction or a
nonspontaneous reaction is a chemical reaction in which the standard change in free energy is positive, and energy is absorbed. Under constant temperature and constant pressure conditions, this means that the change in the standard Gibbs free energy would be positive

for the reaction at standard state (i.e. at standard

pressure (1 bar), and standard concentrations (1 molar of all the reagents).

(3) isoergonic / isothermic reaction

is a reaction in which the temperature
remains (or is kept) constant.

How enzymes work?

(3) Energy change
The energy needed to start a chemical reaction is called the activation energy (EA). The enzyme lowers the activation energy so that the reaction can take place more easily.

How enzymes work?

(2) Molecule geometry
(a) Lock and Key Hypothesis The enzyme had a particular shape into which the substrate(s) fit exactly. The substrate molecule fits into the active site of the enzyme molecule like a key fitting into a lock. This model explains enzyme specificity but fails to explain the stabilisation of the transition state which occurs.

How enzymes work?

(2) Molecule geometry
(b) Induced Fit Theory - The flexible enzyme changes its form to fit the shape of the substrate. - The active site of an enzyme can be modified / change shape as the substrate binds with the enzyme. - This explains enzyme specificity and the stabilisation of the transition state which occurs.

Induced fit theory

Enzyme's shape changes when substrate binds to active site Amino acids are moulded into a precise form to perform catalytic reaction effectively Enzyme wraps around substrate to distort it The bond in the substrate is activated forming ES complex to the point of rupture, releasing the products and the free enzyme binds to a new substrate. Forms an enzyme-substratecomplexfastreaction E + S ES P + E Enzyme is not used up in the reaction (unlike substrates)

COFACTORS
Some enzymes bind with non-protein

groups known as cofactors before they can catalyse a reaction. Enzyme + cofactor = holoenzyme Enzyme cofactor = apoenzyme Inorganic cofactors are metallic ions (Cl-, Mg2+) Organic cofactors are coenzymes (NAD, complex groups vitamins)

COENZYMES
Characteristics: Heat-stable, nonprotein, low molecular weight, organic compound required by some enzymes. Firmly attached to apoenzymes called as prostheticgroupsnondialyzable. Loosely bound to apoenzyme, only coming inclosecontactwithitduringreaction dialyzable.

Classification of Coenzymes
Usually required by oxidoreductases
transferases,isomerases and ligases Classified into: (1) Coenzymes involved in Hydrogen, or Electron, Transfer e.g. NAD, heme (2) Coenzymes involved in transfer of other groups e.g. coenzyme A (carries COOH), biotin (carries CO2)

Metal Ion Activators

Inorganic metal ions are required for the
activity of more than 25% of enzymes. The metal-activated enzymes involves the metal ions loosely attached to the apoenzyme called activators. The metalloenzymes contains metal ions that are firmly attached to the apoenzyme called prosthetic groups.

Role of metal ions in enzyme action

A metal ion (M) forms a complex with an

enzyme (E) and a substrate (S) : 1. Metal bridge complex : E-M-S is formed during the reaction where it acts as a bridge between the enzyme and substrate. 2. Substrate bridge complex : E-S-M is formed where most kinases, ATP act as a bridge between the enzyme and the metal ion.

Role of metal ions in enzyme action

3. Enzyme bridge complex : S-E-M is formed where the enzyme forms a bridge between the substrate and the metal ion.

4. Coenzyme-like action of metal ions : The copper prosthetic group of some oxidases acts as a temporary carrier of electrons from the substrate to O2 to form H2O.

Specificity of Enzymes
As they are highly specific, they may only
catalyze one reaction or a small group of related reactions. This is due to the nature and arrangement of the chemical groups at the catalytic site which allow the enzyme to bind with and activate only one substrate or a small number of structurally related substrates.

Types of specificity
(1) Low Specificity only the linkage should be of the correcttypeenzymeactsonalargenumberof substrates. (2) Group Specificity the linkage and one part of the substrate should be of the correct type--> enzyme acts on a small group of related substrates. (3) Absolute Specificity the linkage and the 2 parts of thesubstrateshouldbeofthecorrecttypeenzyme only acts on one substrate. (4) Stereochemical Specificity if the substrate has stereoisomers, the enzyme only works on one of them.

Importance of Enzyme Specificity

Low specificity of digestive enzymes allows
only a few enzymes to digest all food. High specificity of intracellular enzymes allows metabolic pathways to work and to be regulated efficiently.

ISOENZYMES
Enzymes that catalyze the same reaction but differ in
: (1) Structure ( a.a. sequence) (2) Properties (Km, affinity to substrate, heat, stability, inhibition) (3) Enzymatic activities Usually produced by different cells / compartments of the same cell, in the same species. e.g. lactate dehydrogenase in human body

Lactate dyhydrogenase (LD or LDH)

Enzyme protein of 4 polypeptides chains where
each one of 2 types called H (from heart) and M (muscle). Types: LD1: HHHH (H4) found in cardiac muscle LD2 : HHHM(H3M) in erythrocytes LD3 : HHMM (H2M2) in lungs LD4 : HMMM (HM3) in other tissues LD5 : MMMM (M4) in liver and skeletal muscles.

Lactate dyhydrogenase (LD or LDH)

All the 5 types of isoenzymes are present
in the blood plasma Separation can take place by electrophoresis. LD1 is the fastest and LD5 is the slowest.

Creatine Kinase (CK)

Also known as creatine phosphokinase

(CPK) Protein of 2 polypeptide where each of two types is called M (muscle) and B (brain). Have 3 isoenzymes: CK1 is Ck-BB found in the brain. CK2 is CK-MB in cardiac and skeletal muscles. CK3 is CK-MM in cardiac and skeletal muscles.

Creatine Kinase (CK)

Serum CK levels increase in muscular
dystrophy and after severe muscular exercise in an untrained person where MB fraction is less than 5%. MB fraction more than 5% could lead to acute myocardial infarction. CK-BB levels increase in cases of brain infarction.

Creatine Kinase and CKI

Cell damage

contents spill out

detected in blood

CKtotal 0-220 U/L CKMB 0-5 ng/mL Muscle damage CKtotal elevated CK index (CKI) = CKMB/CKtotal (x 100 for %) Skeletal origin CKI <0.03 (3%) or use CKMM Cardiac origin CKI >0.06 (6%) If 0.03 (3%) > CKI <0.05 (5%) follow with Troponin biomarkers

Muscle Proteins and Diseases

http://www-ermm.cbcu.cam.ac.uk/0200488Xh.htm

In Situ Dystrophin

Normal dystrophin staining

around the rim of muscle fibers
n

Absent dystrophin: Duchenne muscular dystrophy

Left: No staining around the rim of muscle fibers. Right: No staining of most muscle fibers. One "revertant" fiber with dystrophin staining.

http://www.neuro.wustl.edu/neuromuscular/pathol/dmdpath.htm

Western Blots

Western blot of dystrophin from dystrophinopathies.

Lane 3: Normal; Dystrophin has normal size and amount. Lane 1: Becker dystrophy; Dystrophin has reduced abundance but normal
size. Lane 2: Becker dystrophy; Dystrophin has reduced size and abundance. Lane 4: Duchenne dystrophy; Almost no protein is present. Lane 5: Duchenne outlier; Dystrophin has severely reduced abundance.
http://www.neuro.wustl.edu/neuromuscular/pathol/dmdpath.htm

Factors affecting the rate of enzyme-catalyzed reactions

Rate of enzyme-catalyzed reactions is
directly proportional to the concentration of ES complex The rate is affected by: (1)Temperature (2) pH (3)Concentrations of substrate, enzyme, cofactors, inhibitors and allosteric modifiers (4) time

Temperature
As ToC increase, both the enzyme and substrate molecules have more kinetic energy so collide more often, and have sufficient energy to overcome the activation energy. The enzymes have an optimum ToC at which they work fastest. Above opt. ToC, the enzymes will denatured as the H-bonds are broken down and alters the shape of the enzyme and active site. The substrate cannot fit into the active site of enzyme, so, rate of reaction decrease/ stop.

Temperature
Increased Temperature Increases speed of molecularmovement chances of molecular collisionsmoreES complexes At 0 - 42 C, rate of reaction is proportional to temperature. Enzymes have optimum temp. for their action (varies between different enzymes) Above42C, enzyme is denatured due to heavy vibration that break -H bonds Shape is changed / active site can't be used anymore Decreased Temperature Enzymes become less and less active, due to reductions in speed of molecular movement Below freezing point Inactivated, not denatured Regain their function when returning to normal temperature.

 Changes in pH will also

pH

denature the enzyme by changing the shape of the enzyme. Enzymes have an optimum pH at which they work fastest. As the pH decreases from, or increases from the optimum, the acid or base conditions begin to disrupt some of the hydrogen bonds between loops of the protein chains. The active site becomes distorted and substrate can not fit perfectly. Thus, slow down /reduce the rate of reaction.

Changes in pH
Affect attraction between substrate and enzyme and
therefore efficiency of conversion process Ionic bonds can break and change shape / enzyme is denatured Charges on amino acids can change, ES complex cannot form Optimum pH
pH 7 for intracellular enzymes Acidic range (pH 1-6) in the stomach for digestive enzymes (pepsin) Alkaline range (pH 8-14) in oral cavities (amylase)

pH measures the conc. of H+ ions

higher conc. will give a lower pH

Concentration of Enzyme [E]

As the [E] increases, the rate of the
reaction increases linearly, because there are more enzyme molecules available to catalyse the reaction. At very high [E], the substrate concentration may become ratelimiting, so the rate stops increasing. As large excess substrate is maintained, the rate of reaction increase as [enzyme] increases.
Linear relationship

Enzyme Concentration
is proportional to rate
of reaction, provided other conditions are constant. show a straight line.

Substrate Concentration
- As the [substrate] increases,

the rate increases because more substrate molecules can collide with enzyme molecules, so more reactions will take place. - At higher concentrations, the enzyme molecules become saturated with substrate, so there are few free enzyme molecules, so adding more substrate doesn't make much difference.

Substrate Concentration
is proportional to rate
of reaction until there are more substrates than enzymes present, at which the maximum rate of reaction, Vmax is reached. shows a curve that becomes constant.

Michaelis constantorKm
The relation between the initial velocity of the
reaction,v and the substrate concentration, [S], can be expressed by the Michelis-Menten equation which gives a hyperbolic relation. The substrate concentration that gives Vmax is knownastheMichaelis constantorKm.

Enzymes
k1
S E S E

k2

P
5 4

k-1

Vma x[S] v Km [S]

0.5Vmax

20 15
v, mol/min

-Km, Vmax
v, mol/min

10 5 0 -5 -10 -50

3 2 1 0

Km
0 10 20 30 [S], mM 40 50

-30

-10 10 [S], mM

30

50

Eric Niederhoffer SIU-SOM

Significance of Km
:
- depends on pH, ionic strength and temperature but NOT enzyme concentration. - Indicates the affinity between enzyme and the substrate-- the lower Km, the greater affinity. An inhibitor interferes with the binding increases the Km as higher substrate conc. is needed to reach the velocity. - used to characterize isoenzymes as they have different Km.

Time
As reaction proceeds, the velocity of reaction decrease due to: (1) decreased conc. of substrate (2) increased conc. of products which tend to reverse the reaction (3) denaturation of enzyme.

** Take note
Must always determine the initial velocity of
reaction when studying the factors affecting the rate of enzyme-catalyzed reactions. That the amount of substrate converted to products continues to increase provided that : (1) the equilibrium point of reaction is not reached and (2) the enzyme is not inactivated.

Inhibition of enzyme
Inhibit (slow down) the activity of enzyme which reduce the rate of reaction. Inhibition may be reversible, or irreversible. Reversible inhibitors generally bind to enzymes non-covalently. Irreversible inhibitors are generally covalently bound to the enzyme, thus permanently disabling the enzyme. There are two types of inhibition: (1) Competitive (active site-directed) (2) Non-competitive (non-active site-directed)

Competitive Inhibition
Also known as active sitedirected inhibition. A competitive inhibitor molecule has a similar structure to the normal substrate molecule, and it can fit into the active site of the enzyme. When both substrate and inhibitor molecules are present, they `compete with each other for the active site, so the reaction (catalytic action of the enzyme) is slower.

Competitive Inhibitors
Form an enzyme-inhibitor complex (E-I
complex) leading towards the decrease of E-S complex, thus, inhibiting the activity of the enzyme. Also known as substrate analogue inhibitors or metabolic antagonists

Degree of inhibition
depends on: (1) the ratio of the conc. of the inhibitor and the
substrate. if more substrate is added at a fixed conc. of the inhibitor, the degree of inhibition decreases. (2) the relative affinity of the substrate and the inhibitor for the enzyme. if the inhibitor binds tightly to the enzyme, the degree of inhibition will be greater than if it binds loosely.

Competitive Inhibitors
Compete with substrate for active site. Shape similar to substrates / prevents access when
bonded. Can slow down a metabolic pathway. EXAMPLE: Methanol Poisoning Methanol CH3OH is a competitive inhibitor . CH3OH can bind to dehydrogenase whose true substrate is C2H5OH . A person who has accidentally swallowed methanol is treated by being given large doses of C2H5OH. C2H5OH competes with CH3OH for the active site.

Competitive Inhibition

Non-competitive Inhibition
Also known as non-active
site-directed inhibition. A non-competitive inhibitor molecule is quite different in structure from substrate molecule . It binds to another part of the enzyme molecule, changing the shape of the whole enzyme, including the active site, so that it can no longer bind substrate molecules.

Non-competitive Inhibitors
Chemical does not have to resemble the
substrate. Binds to enzyme other than at active site. This changes the enzyme's active site and prevents access to it.

Non-competitive Inhibition

Irreversible Inhibition
Chemical permanently binds to the enzyme or
massively denatures the enzyme. Nerve gas permanently blocks pathways involved in nerve message transmission, resulting in death. Penicillin, the first of "wonder drug" antibiotics, permanently blocks pathways that certain bacteria use to assemble their cell wall component (peptidoglycan.)

Enzymatic pathway
Formed as a result of the common occurrence of a series

of dependent chemical reactions. Can be located adjacent to each other (in an organelle or in the membrane of an organelle), thus speeding the reaction process. The end product depends on the successful completion of few reactions, each mediated by a specific enzyme. The substrate interacts with the enzyme-1, E1 to form products. One of these products becomes the substrate for the next enzyme, and continues through several more enzymes. When a higher concentration of final product begins to build up, it "feeds back" to the first enzyme and reacts at the allosteric site. The enzyme changes shape so that the substrate does not fit into the active site, and no more reactions can take place.

Allosteric Interaction in feedback inhibition

Also considered as a non-competitive inhibition Inhibitor bind to other site of enzyme (allosteric site) and alters the shape of enzyme. Substrate cannot fit into the active site. Thus, stop reaction.

Allosteric Interactions
Involve the feedback inhibition which allow enzyme to be temporarily inactivated when one of the products, either an end or near-end product acts as an inhibitor to block or shut the pathway. It changes the shape of the enzyme, inactivating it while the inhibitor is still bound to the allosteric site of the enzyme.

End-product inhibition
Metabolic reactions are multi-stepped, each
controlled by a single enzyme End-products accumulate within the cell and stop the reaction when sufficient product is made This is achieved by non-competitive inhibition by the end-product. The enzyme early in the reaction pathway is inhibited by the end-product. This process also known as feedback inhibition.

Feedback Inhibition
If the product of a series of enzymatic reactions begins to
accumulate within the cell, it may specifically inhibit the action of the first enzyme involved in its synthesis (red bar). Thus, further production of the enzyme is halted.