enzyme activity and characterization.pdf

8/10/2019 Enzyme Activity and Characterization.pdf

1/14


2/14

ENZYMESARE:

Proteins (note that recent developments indicate that both RNA and

antibodies may have catalytic activity, these are called ribozymes,

and catalytic antibodies or abzymes, respectively)

Biological catalysts, critical components of cell metabolism &

biological processes

Very efficient catalysts

Like other catalysts, enzymes do not alter the position of equilibrium between

substrates and products. However, unlike normal chemical reactions, enzymesare saturable. This means as more substrate is added, the reaction rate will

increase, because more active sites become occupied.
http://upload.wikimedia.org/wikipedia/commons/0/09/KinEnzymo%28en%29.svghttp://upload.wikimedia.org/wikipedia/commons/0/09/KinEnzymo%28en%29.svghttp://upload.wikimedia.org/wikipedia/commons/0/09/KinEnzymo%28en%29.svg


3/14

Very specific catalysts

Reduce G for reaction (by binding the transition state)

Subject to regulatory control of various sorts

Carry out catalysis in a special region of the molecule, the

active-site

Exhibit special kinetics
http://en.wikipedia.org/wiki/Image:Activation2_updated.svg


4/14

All enzymes are proteins, with the exception of some small

catalytic RNAs and RNA/protein complexes.

MW's range from 104 to 106 daltons.

May be single polypeptide chains, or oligomers of several

subunits (most commonly oligomers are dimers, or tetramers,

some multienzyme complexes as many as 48 protomers).

May have more than one activity associated with the same protein

(i. e. there are some large enzymes which catalyze more than onereaction (frequently successive steps in a metabolic pathway).

Often contain a prosthet ic group (or cofactor): Typical examples

are: metal ions, heme, Fe-S clusters, coenzymes (e.g. NADH, FAD,

FMN, PLP) .

Coenzymes usually are vitamins, or derived from vitamins, and act

as carriers (e.g. of H, e-, CO2).

Enzymes are usually named after their substrate by adding ase,

e. g. protease (proteinase), esterase,

-glucosidase, alcohol

dehydrogenase,

-lactamase.


5/14

Biolo gical catalysts:

Catalysts speed the rate of attainment of equilibrium by

lowering the energy barrier between substrate andproducts. In other words a catalyst will increase the rate

of a reaction but not affect the position of the reaction

equilibrium. The catalyst is not used up in the reaction but

is regenerated.

Specif ic i ty:

This is the second unique feature of enzymes as

catalysts; they are very specific. A given enzyme will only

catalyze one type of reaction for one type of compound, in

some cases for only one compound. They are also verystereospecif ic ,and produce no by-products.


6/14

Free Energy of Activation

Enzymes act as catalysts because they lower the free energy of activation

(G) for the reaction. They do this by a combination of raising the ground

state G of the substrate and lowering the G of the transition state (TS)

for the reaction, thereby decreasing the barrier for reaction to occur. Thepresence of the enzyme leads to a new (different) reaction pathway than

for the uncatalyzed reaction. As we will see, the major way in which

enzymes bring about their great rate enhancements is by tight binding of

the TS.

The height of the energy barrier is the free energy of activation.


7/14

Enzyme assaysare laboratory methods for measuring enzymatic

activity. They are vital for the study of enzyme kinetics and enzyme

inhibition.

Amounts of enzymes can either be expressed as molar amounts, as with

any other chemical, or measured in terms of activity, in enzyme units.

Enzyme activity = moles of substrate converted per unit time = rate

reaction volume.

Enzyme activity is a measure of the quantity of active enzyme present and

is thus dependent on conditions, which should be specified.

The SI unit is the katal, 1 katal = 1 mol s-1, but this is an excessively

large unit.

A more practical and commonly-used value is 1 enzyme unit (EU)= 1

mol min-1 ( = micro, x 10-6). 1 U corresponds to 16.67 nanokatals.

The specific activityof an enzyme is another common unit. This is the

activity of an enzyme per milligram of total protein (expressed in mol min -

1mg-1). Specific activity gives a measurement of the purity of the enzyme.


8/14

Enzyme Assays

All enzyme assays measure either the consumption of substrate or

production of product over time.

Methode:

Spectrophotometric assays(colorimetric assays): the course of the

reaction by measuring a change in how much light the assay solution

absorbs. If this light is in the visible region you can actually see a change

in the color of the assay, these are called

Diagram of a single-beam UV/vis spectrophotometer
http://en.wikipedia.org/wiki/Image:UV-vis.png


9/14

Fluorimetric assays

Fluorescence is when a molecule emits light of one wavelength after

absorbing light of a different wavelength.

Fluorometric assays use a difference in the fluorescence of substrate

from product to measure the enzyme reaction. These assays are in

general much more sensitive than spectrophotometric assays, but can

suffer from interference caused by impurities and the instability of

many fluorescent compounds when exposed to light.

An example of these assays is again the use of the nucleotide

coenzymes NADH and NADPH. The reduced forms are fluorescent

and the oxidised forms non-fluorescent. Oxidation reactions can

therefore be followed by a decrease in fluorescence and reductionreactions by an increase.

Synthetic substrates that release a fluorescent dye in an enzyme-

catalyzed reaction are also available, such as 4-methylumbelliferyl--

D-glucuronide for assaying -galactosidase.
http://en.wikipedia.org/wiki/%CE%92-galactosidasehttp://en.wikipedia.org/wiki/%CE%92-galactosidasehttp://en.wikipedia.org/wiki/%CE%92-galactosidasehttp://en.wikipedia.org/wiki/%CE%92-galactosidasehttp://en.wikipedia.org/wiki/%CE%92-galactosidase


10/14

Factors to control in assays

Salt Concentration: Most enzymes can not tolerate extremely high salt

concentrations. The ions interfere with the weak ionic bonds of proteins.Typical enzymes are active in salt concentrations of 1-500 mM.

Effects of Temperature: All enzymes work within a range of temperature

specific to the organism. Increases in temperature generally lead to

increases in reaction rates. There is a limit to the increase because higher

temperatures lead to a sharp decrease in reaction rates. This is due to thedenaturating (alteration) of protein structure resulting from the breakdown of

the weak ionic and hydrogen bonding that stabilize the three dimensional

structure of the enzyme. However, the idea of an "optimum" rate of an

enzyme reaction is misleading, as the rate observed at any temperature isthe product of two rates, the reaction rate and the denaturation rate.


11/14

Effects of pH: Most enzymes are sensitive to pH and have specific

ranges of activity. All have an optimum pH. The pH can stop enzyme

activity by denaturating (altering) the three dimensional shape of the

enzyme by breaking ionic, and hydrogen bonds.

Substrate Saturation: Increasing the substrate concentration

increases the rate of reaction (enzyme activity). However, enzyme

saturation limits reaction rates. An enzyme is saturated when the

active sites of all the molecules are occupied most of the time. At the

saturation point, the reaction will not speed up, no matter how much

additional substrate is added. The graph of the reaction rate will

plateau.
http://en.wikipedia.org/wiki/Image:Michaelis-Menten_saturation_curve_of_an_enzyme_reaction.svg


12/14

Amylase

Amylaseis the name given to glycoside hydrolase enzymes that

break down starch into maltose molecules. They all act on -1,4-

glycosidic bonds.

Starch is a mixture of amylose and amylopectin (usually in 20:80

or 30:70 ratios). These are both complex carbohydrate polymersof glucose

Amylose structure

Amylopectin structure
http://en.wikipedia.org/wiki/Image:Amylopectin.svghttp://en.wikipedia.org/wiki/Image:Amylose.svghttp://upload.wikimedia.org/wikipedia/en/4/4f/Hydrolase_mech.gifhttp://en.wikipedia.org/wiki/Image:Hydrolase_mech.gifhttp://en.wikipedia.org/wiki/Image:Hydrolase_mech.gif


13/14

Classification-Amylase (EC 3.2.1.1)

alternate names: 1,4--D-glucan glucanohydrolase; glycogenase).

The -amylases are calcium metalloenzymes, completely unable to function in theabsence of calcium. By acting at random locations along the starch chain, -

amylase breaks down long-chain carbohydrates, ultimately yielding maltotriose

and maltosefrom amylose, or maltose, glucoseand "limit dextrin"from

amylopectin. Because it can act anywhere on the substrate, -amylase tends to be

faster-acting than -amylase.

-Amylase (EC 3.2.1.2)

alternate names: 1,4--D-glucan maltohydrolase; glycogenase; saccharogen

amylase.

Another form of amylase, -amylase is also synthesized by bacteria, fungi, and

plants.

Working from the non-reducing end, -amylase catalyzes the hydrolysis of the

second -1,4 glycosidic bond, cleaving off two glucose units (maltose) at a time. is

key to the production of malt. Many microbesalso produce amylase to degrade

extracellular starches.
http://en.wikipedia.org/wiki/Maltotriosehttp://en.wikipedia.org/wiki/Maltosehttp://en.wikipedia.org/wiki/Amylosehttp://en.wikipedia.org/wiki/Glucosehttp://en.wikipedia.org/wiki/Dextrinhttp://en.wikipedia.org/wiki/Amylopectinhttp://en.wikipedia.org/wiki/Substrate_%28biochemistry%29http://en.wikipedia.org/wiki/Bacteriahttp://en.wikipedia.org/wiki/Fungihttp://en.wikipedia.org/wiki/Planthttp://en.wikipedia.org/wiki/Maltosehttp://en.wikipedia.org/wiki/Malthttp://en.wikipedia.org/wiki/Microbehttp://en.wikipedia.org/wiki/Microbehttp://en.wikipedia.org/wiki/Malthttp://en.wikipedia.org/wiki/Maltosehttp://en.wikipedia.org/wiki/Planthttp://en.wikipedia.org/wiki/Fungihttp://en.wikipedia.org/wiki/Bacteriahttp://en.wikipedia.org/wiki/Substrate_%28biochemistry%29http://en.wikipedia.org/wiki/Amylopectinhttp://en.wikipedia.org/wiki/Dextrinhttp://en.wikipedia.org/wiki/Glucosehttp://en.wikipedia.org/wiki/Amylosehttp://en.wikipedia.org/wiki/Maltosehttp://en.wikipedia.org/wiki/Maltotriose


14/14

-Amylase (EC 3.2.1.3 )

alternative names: Glucan 1,4--glucosidase; amyloglucosidase; Exo-

1,4--glucosidase; glucoamylase; lysosomal -glucosidase; 1,4--D-

glucan glucohydrolase) In addition to cleaving the last (1-4)glycosidiclinkages at the nonreducing end of amylose and amylopectin, yielding

glucose, -amylase will cleave (1-6) glycosidic linkages.

Pullulanase (EC 3.2.1.41) is also known as pullulan-6-glucanohydrolase

(Debranching enzyme). Its substrate, pullulan, is regarded as a chain of

maltotriose units linked by alpha-1,6-glycosidic bonds. Pullulanase willhydrolytically cleave pullulan (alpha-glucan polysaccharides).

Pullulanaseis a specific kind of , an amylolytic exoenzyme, that degradespullulana polysaccharide polymerconsisting of maltotriose units, also known as -

1,4- ;-1,6-glucan.

It is produced as an extracellular, cell surface-anchored lipoprotein by Gram-negative bacteria of the genus Klebsiella. Type I pullulanases specifically

attack -1,6 linkages, while type II pullulanases are also able to hydrolyse -

1,4 linkages. It is also produced by some other bacteria and archaea.

Pullulanase is used as a detergent in biotechnology.

enzyme activity and characterization.pdf

Documents