enzyme - university of babylon
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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
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).
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.
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)
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