Enzyme

Abdulhussien Mahdi Aljebory

College of pharmacy

University of Babylon

What Are Enzymes?

• Most enzymes are Proteins (tertiary and quaternary structures)

• Act as Catalyst to accelerates a reaction

• Not permanently changed in the process

• Are specific for what they will catalyze• Are Reusable• End in –ase-Sucrase-Lactase-Maltase

How do enzymes Work?

Enzymes work by weakening bonds which lowers activation energy.

Enzyme-Substrate Complex

The substance (reactant) an enzyme acts on is the substrate

Substrate Joins Enzyme

FreeEnergy

Progress of the reaction

Reactants

Products

Free energy of activation

With out Enzyme With Enzyme

❖ Active Site

• A restricted region of an enzyme molecule which binds to the substrate.

❖Induced Fit:

• A change in the shape of an enzyme’s

active site Induced by the substrate.

EnzymeSubstrate

Active Site

What Affects Enzyme Activity?

• Three factors:

1. Environmental Conditions

2. Cofactors and Coenzymes

3. Enzyme Inhibitors

1. Environmental Conditions

1. Extreme Temperature are the most dangerous

- high temps may denature (unfold) the enzyme.

2. pH (most like 6 - 8 pH near neutral)

3. Ionic concentration (salt ions)

Optimum pH Values

Enzymes in• the body have an optimum pH of about 7.4.• certain organs operate at lower and higher optimum pH values.

Substrate ConcentrationAs substrate concentration increases,

• the rate of reaction increases (at constant enzyme concentration).

• the enzyme eventually becomes saturated, giving maximum activity.

Quiz Sucrase has an optimum temperature of 37 °C and an optimum pH of 6.2. Determine the effect of the following on its rate of reaction.

1) no change 2) increase 3) decrease

A. Increasing the concentration of sucrose

B. Changing the pH to 4

C. Running the reaction at 70 °C

2. Cofactors and Coenzymes

• Inorganic substances (zinc, iron) and vitamins(respectively) are sometimes need for proper enzymatic activity.

• Example:Iron must be present in the quaternary structure -

hemoglobin in order for it to pick up oxygen.

Enzyme Inhibition

Inhibitors

• are molecules that cause a loss of catalytic activity.

• prevent substrates from fitting into the active sites.

E + S ES E + P

E + I EI no P

Two examples of Enzyme Inhibitors

A- competitive inhibitor

• has a structure that is similar to that of the substrate.

• competes with the substrate for the active site.

• has its effect reversed by increasing substrate concentration.

Enzyme

Competitive inhibitor

Substrate

B- noncompetitive inhibitor

• has a structure that is much different than the substrate.

• distorts the shape of the enzyme, which alters the shape of the active site.

• prevents the binding of the substrate.

• cannot have its effect reversed by adding more substrate.

Enzyme

active sitealtered

NoncompetitiveInhibitor

Substrate

Mechaelis Menton kinetics

Plotting Vi as a function of [S], we find that

➢At low values of [S], the initial velocity,Vi, rises almost linearly with increasing [S].

➢But as [S] increases, the gains in Vi level off (forming a rectangular hyperbola).

➢ The asymptote represents the maximum velocity of the reaction, designated Vmax

➢ The substrate concentration that produces a Vi that is one-half of Vmax is designated the Michaelis-Menten constant, Km(named after the scientists who developed the study of enzyme kinetics).

➢Km is (roughly) an inverse measure of the affinity or strength of binding between the enzyme and its substrate. The lower the Km, the greater the affinity (so the lower the concentration of substrate needed to achieve a given rate).

Plotting out our data it might look like this.

Lineweaver-Burke plot

Plotting the reciprocals of the same data points yields a "double-reciprocal" or Lineweaver-Burk plot. This provides a more precise way to determine Vmax and Km. Vmax is determined by the point where the line crosses the 1/Vi = 0 axis (so the [S] is infinite). Note that the magnitude represented by the data points in this plot decrease from lower left to upper right. Km equals Vmax times the slope of line. This is easily determined from the intercept on the X axis.

Competitive/noncompetitive inhibitor

Effect of inhibitors

The KM widely varies among different enzymes

The KM

can be expressed as:

1

2

1

2

1

1 KKk

k

k

k

k

ksM +=+= −

As Ks decreases, the affinity for the substrate increases. The KM can be a measure for substrate affinity if k2<k-1

• Michaelis constants: have been determined for many of the commonly used enzymes. The size of Km tells us several things about a particular enzyme.

1. A small Km indicates that the enzyme requires only a small amount of substrate to become saturated. Hence, the maximum velocity is reached at relatively low substrate concentrations.

2. A large Km indicates the need for high substrate concentrations to achieve maximum reaction velocity.

3. The substrate with the lowest Km upon which the enzyme acts as a catalyst is frequently assumed to be enzyme's natural substrate, though this is not true for all enzymes.

Lineweaver-Burk plot: slope = KM/Vmax,

1/vo intercept is equal to 1/Vmax

the extrapolated x intercept is equal to -1/KM

For small errors in at low [S] leads to large errors in 1/vo

Tmax

E

V=catk

kcat is how many reactions an enzyme can catalyze per second

The turnover number

For Michaelis -Menton kinetics k2= kcat

When [S] << KM very little ES is formed and [E] = [E]T

and

Kcat/KM is a measure of catalytic efficiency

What is catalytic perfection?

When k2>>k-1 or the ratio21

21

kk

kk

+−

is maximum

Then1

MKk

kcat =Or when every substrate that hits the enzyme causes a reaction totake place. This is catalytic perfection.

Diffusion-controlled limit- diffusion rate of a substrate is in the range of 108 to 109 M-1s-1. An enzyme lowers the transition state so there is no activation energy and the catalyzed rate is as fast as molecules collide.

Quiz -2

Identify each description as an inhibitor that is

1) competitive or 2) noncompetitive.

A. Increasing substrate reverses inhibition.B. Binds to enzyme surface, but not to the active site.C. Structure is similar to substrate.D. Inhibition is not reversed by adding more substrate.

solution

Identify each description as an inhibitor that is

1) competitive or 2) noncompetitive.

A. 1 Increasing substrate reverses inhibition.

B. 2 Binds to enzyme surface, but not to the active site.

C. 1 Structure is similar to substrate.

D. 2 Inhibition is not reversed by adding more substrate.

Measurement of ENZYME ACTIVITY

• The enzyme activity can be defined as the number of moles ofsubstrate converted per unit time.

• Enzyme activity = moles of substrate converted per unit time

• = rate × reaction volume.

• The SI unit is the katal, 1 katal = 1 mol s−1

• A more practical and commonly used value is 1 enzyme unit (U) =1 μmol min−1

TYPES OF ENZYME ASSAYS

CONTINUOUS ASSAYS

• With continuous assays, one canmeasure the linearity of the assaywhich can be used to conduct afixed-timed assay

• A few methods arespectrophotoometric, fluorometric,calorimetric and chemi-luminescent.

DISCONTINUOUS ASSAYS

• Discontinuous assays are whensamples are taken from anenzyme reaction at intervals andthe amount of productproduction or substrateconsumption is measured inthese samples.

• The discontinuous assays areradiometric andchromatographic.

Measurement Of Enzymatic Activity By

Spectroscopy

• Spectrophotometry is the measureable analysis technique using the

electromagnetic spectra. It deals with the ranges of wavelengths such as

near ultraviolet, near infrared and visible light

• Activity of the enzyme, the following spectroscopic techniques are used:

Fluorescence spectroscopy, UV/VIS Spectroscopy, Spectrophotometric

Assays, and Infrared spectroscopy.

Fluorescence spectroscopy

• Fluorescence spectroscopy reveals the existence of ES complexes and

what they are made of.

• A compound is exposed to UV-light which excites certain molecules and

causes them to emit light at a lower wavelength, which is in the visible

light range.

• The fluorescence of the substrate is measured and compared to the

fluorescence of the product, and in the difference of the two

measurements, enzymatic activity is measured

Infrared Spectroscopy

• In enzyme-substrate complexes, there are well-organized binding

modes, which is quantifiable using infrared methods.

• In analyzing infrared data, it is possible to identify binding modes and

heterogeneity of ES complexes.

Ultraviolet-visible Spectroscopy

• It is a commonly used spectrophotometric assay that examines

photons in the UV-visible region.

• It is mainly used to determine the amount of a highly-conjugated

organic compound or enzyme contained in a specific solution.

• It is complimentary to fluorescence spectroscopy, as fluorescence

spectroscopy deals with transitions from the excited state to the

ground state, ultraviolet-visible spectroscopy deals with transitions

from the ground state to the excited state.

• The device, spectrophotometer, measures thetransmittance of light through a sample

The equation used to calculate the transmittance is

A=-log(%T)

Here, A= absorbance

T= I/Io

I=the intensity of light passingthrough the sample

Io=the initial intensity of the lightbefore it is transmitted

through the sample

Radiometric assay

• It measures the incorporation of radioactivity into substrates or its release

from substrates.

• The radioactive isotopes most frequently used in these assays

are 14C, 32P, 35S and 125I.

• These assays are both extremely sensitive and specific.

• They are often the only way of measuring a specific reaction in crude

extracts (the complex mixtures of enzymes produced when you lyse cells).

Chromatographic assay

• It measures product formation by separating the

reaction mixture into its components

by chromatography.

• This is usually done by high-performance liquid

chromatography (HPLC), but can also use the simpler

technique of thin layer chromatography.

• Although this approach can need a lot of material, its

sensitivity can be increased by labelling the

substrates/products with a radioactive or fluorescent tag, by

switching protocols to improved chromatographic

instruments (e.g. ultra-high pressure liquid chromatography)

that operate at pump pressure a few-fold higher than HPLC

instruments.

Specificity of Enzymes

• 1. Absolute specificity - the enzyme will catalyze only one reaction.

• 2. Group specificity - the enzyme will act only on molecules that have

specific functional groups, such as amino, phosphate and methyl groups.

• 3. Linkage specificity - the enzyme will act on a particular type of chemical

bond regardless of the rest of the molecular structure.

• 4. Stereochemical specificity - the enzyme will act on a particular steric or

optical isomer.

Naming and Classification

Except for some of the originally studied enzymes such as pepsin, rennin,

and trypsin, most enzyme names end in "ase". The International Union of

Biochemistry (I.U.B.) initiated standards of enzyme nomenclature which

recommend that enzyme names indicate both the substrate acted upon

and the type of reaction catalyzed. Under this system, the enzyme

uricase is called urate: O2 oxidoreductase, while the enzyme glutamic

oxaloacetic transaminase (GOT) is called L-aspartate: 2-oxoglutarate

aminotransferase.

Enzymes can be classified by the kind of chemical reaction catalyzed.

• I. Addition or removal of water

• A. Hydrolases - these include esterases, carbohydrases, nucleases, deaminases,

• amidases, and proteases

• B. Hydrases such as fumarase, enolase, aconitase and carbonic anhydrase

• II. Transfer of electrons

• A. Oxidases

• B. Dehydrogenases

• III. Transfer of a radical• A. Transglycosidases - of monosaccharides• B. Transphosphorylases and phosphomutases - of a phosphate group• C. Transaminases - of amino group• D. Transmethylases - of a methyl group• E. Transacetylases - of an acetyl group

• IV. Splitting or forming a C-C bond• A. Desmolases• V. Changing geometry or structure of a molecule• A. Isomerases• VI. Joining two molecules through hydrolysis of pyrophosphate bond in ATP or

other• tri-phosphate• A. Ligases

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