ENZYMES ARE:

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, enzymes
are saturable. This means as more substrate is added, the reaction rate will
increase, because more active sites become occupied.

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

 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 one
reaction (frequently successive steps in a metabolic pathway).

Often contain a prosthetic 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.

Biological catalysts:
Catalysts speed the rate of attainment of equilibrium by
lowering the energy barrier between substrate and
products. 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.
Specificity:
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 very
stereospecific, and produce no by-products.

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. The
presence 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.

Enzyme assays are 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 activity of an enzyme is another common unit. This is the
activity of an enzyme per milligram of total protein (expressed in mol min1mg-1). Specific activity gives a measurement of the purity of the enzyme.

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

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 reduction
reactions by an increase.
Synthetic substrates that release a fluorescent dye in an enzymecatalyzed reaction are also available, such as 4-methylumbelliferyl-D-glucuronide for assaying -galactosidase.

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 the
denaturating (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 is
the product of two rates, the reaction rate and the denaturation rate.

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.

Amylase
Amylase is the name given to glycoside hydrolase enzymes that
break down starch into maltose molecules. They all act on -1,4glycosidic bonds.

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

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

Amylose structure

Amylopectin structure

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 the
absence of calcium. By acting at random locations along the starch chain, amylase breaks down long-chain carbohydrates, ultimately yielding maltotriose
and maltose from amylose, or maltose, glucose and "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 microbes also produce amylase to degrade
extracellular starches.

-Amylase (EC 3.2.1.3 )

alternative names: Glucan 1,4--glucosidase; amyloglucosidase; Exo1,4--glucosidase; glucoamylase; lysosomal -glucosidase; 1,4--Dglucan glucohydrolase) In addition to cleaving the last (1-4)glycosidic
linkages 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 will
hydrolytically cleave pullulan (alpha-glucan polysaccharides).
Pullulanase is a specific kind of , an amylolytic exoenzyme, that degrades
pullulana polysaccharide polymerconsisting of maltotriose units, also known as 1,4- ;-1,6-glucan.
It is produced as an extracellular, cell surface-anchored lipoprotein by Gramnegative 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.