PAMANTASAN NG LUNGSOD NG MAYNILA

College of Engineering and Technology

Chemical Engineering Department
CHE 512 Biochemical Engineering


A
WRITTEN
REPORT
ENZYME AND ENZYME KINETICS

Submitted by:
BESA, Rose Lynn Anne
DARANCIANG, Erish
DECAPIA, Handrey
LUDOVICE, Bianca Jamila
BSChE 5

Submitted to:
Engr. Elaine G. Mission

Page 2 of 37


Contents
ENZYME ......................................................................................................................................................... 3
Brief History of Enzyme ............................................................................................................................. 3
Enzyme Classifcaton and Nomenclature ................................................................................................. 4
Common Enzymes ..................................................................................................................................... 9
pH of some Enzymes ............................................................................................................................... 10
Industrial Applicatons ............................................................................................................................ 11
Factors afectng Enzyme Actvity ........................................................................................................... 13
SIMPLE ENZYME KINETICS........................................................................................................................... 16
Introducton to Enzyme Kinetcs ............................................................................................................. 16
Rate of Reacton .................................................................................................................................. 16
Formaton of Enzyme-Substrate Complex ........................................................................................... 18
Lock-and-Key Theory ........................................................................................................................... 18
Induced-Fit Model ............................................................................................................................... 18
Michaelis-Menten Kinetc Model ............................................................................................................ 19
Biographies of Michaelis and Menten ................................................................................................ 22
Derivaton of Rate Equaton (Michaelis-Menten equaton) ................................................................ 23
Biographies of Lineweaver and Burk ................................................................................................... 25
Graphical Analysis ............................................................................................................................... 26
ENZYME REACTOR WITH SIMPLE KINETICS ................................................................................................. 31
General Types of Bioreactors .................................................................................................................. 32
Batch Strred-Tank Reactor .................................................................................................................. 32
Plug-Flow Reactor ............................................................................................................................... 33
Contnuous Strred-Tank Reactor ........................................................................................................ 34
ENZYME INHIBITION ................................................................................................................................... 36
Bibliography ................................................................................................................................................ 37



Page 3 of 37

ENZYME

Enzymes are biological catalysts. They increase the rate of chemical reactons taking place within living
cells without themselves sufering any overall change. The reactants of enzyme-catalyzed reactons are
called substrates. Each enzyme is quite specifc in character, actng on a partcular substrate or substrates
to produce a partcular product.

All known enzymes are proteins. They therefore consist of one or more polypeptde chains and display
propertes that are typical of proteins. Some enzymes require small non-protein molecules, known as
cofactors, in order to functon as catalysts.

Enzymes difer from chemical catalysts in several important ways:
1. Enzyme-catalyzed reactons are at least several orders of magnitude faster than chemically-
catalyzed reactons. When compared to the corresponding unanalyzed reactons, enzymes
typically enhance the rates by 10
6
to 10
13
tmes.
2. Enzymes have far greater reacton specifcity than chemically-catalyzed reactons and they rarely
form byproducts.
3. Enzymes catalyze reactons under comparatvely mild reacton conditons, such as temperatures
below 100C, atmospheric pressure and pH around neutral. Conversely, high temperatures and
pressures and extremes of pH are ofen necessary in chemical catalysis.

Brief History of Enzyme

1833 The actve agent breaking down the sugar was partally isolated and given the name
distase (now known as amylase). A litle later, a substance which digested dietary protein
was extracted from gastric juice and called pepsin. These and other actve preparatons
were given the general name ferments. Justus von Liebig recognized that these ferments
could be non-living materials obtained from living cells, but Louis Pasteur stll maintained
that ferments must contain living material.

1878 The term ferment was gradually replaced by the name enzyme which is proposed by
Wilhelm Kuhne. It comes from a Greek enzume, meaning in yeast.

1897 Brothers Eduard and Hans Buchner showed that sugar fermentaton could take place
when a yeast cell extract was added even though no living cells were present.

1926 James Sumner crystallized urease from jack-bean extracts and in the next few years, many
other enzymes were purifed and crystallized.

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Enzyme Classification and Nomenclature

Enzymes are classifed according to the report of the Nomenclature Commitee appointed by the
Internatonal Union of Biochemistry (1984). This enzyme commission assigned each enzyme a
recommended name and a four-part distnguishing number.

The frst Enzyme Commission, in its report in 1961, devised a system for classifcaton of enzymes that also
serves as a basis for assigning code numbers to them. These code numbers, prefxed by EC, which are now
widely in use, contain four elements separated by points, with the following meaning:
1. The frst number shows to which of the six main classes the enzyme belongs,
2. The second fgure indicates the type of group involved in the reacton,
3. The third fgure denotes the reacton by indicatng substrate on which the group acts,
4. The fourth fgure is the serial number of the enzyme in its sub-subclass.



Class 1. Oxidoreductases

To this class belong all enzymes catalysing redox reactons. The substrate that is oxidized is regarded as
hydrogen donor. The systematc name is based on donor:acceptor oxidoreductase. The common name will
be dehydrogenase, wherever this is possible; as an alternatve, reductase can be used. Oxidase is only used
in cases where O2 is the acceptor.

2nd EC digit: indicates group in the hydrogen donor (substrate oxidized)



The six main classes of enzymes
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3rd EC digit: indicates type of acceptor involved


Example:
Code number: EC 1.1.1.27
Systematc name: (S)-lactate: NAD
+
oxidoreductase
Common name: Lactate dehydrogenase


Class 2. Transferases

Transferases are enzymes transferring a group, e.g. a methyl group or a glycosyl group, from one
compound (generally regarded as donor) to another compound (generally regarded as acceptor). The
systematc names are formed according to the scheme donor:acceptor group transferase. The common
names are normally formed according to acceptor group transferase or donor group transferase. In many
cases, the donor is a cofactor (coenzyme) charged with the group to be transferred.

2nd EC digit: indicates group transferred


3rd EC digit: further informaton on group transferred

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Example:
Code number: EC 2.7.1.1
Systematc name: ATP: D-hexose-6-phosphotransferase
Common name: Hexokinase


Class 3. Hydrolases

These enzymes catalyze the hydrolytc cleavage of C-O, C-N, C-C and some other bonds, including
phosphoric anhydride bonds. Although the systematc name always includes hydrolase, the common name
is, in many cases, formed by the name of the substrate with the sufx -ase. It is understood that the name
of the substrate with this sufx means a hydrolytc enzyme.

2nd EC digit: indicates nature of bond hydrolysed


3rd EC digit: indicates nature of substrate


Example:
Code number: EC 3.1.3.1
Systematc name: Orthophosphoric monoester phosphohydrolase
Common name: Alkaline phosphatase



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Class 4. Lyases

Lyases are enzymes cleaving C-C, C-O, C-N, and other bonds by eliminaton, leaving double bonds or rings,
or conversely adding groups to double bonds. The systematc name is formed according to the patern
substrate group-lyase. The hyphen is an important part of the name, and to avoid confusion should not be
omited, e.g. hydro-lyase not 'hydrolyase'. In the common names, expressions like decarboxylase, aldolase,
dehydratase (in case of eliminaton of CO2, aldehyde, or water) are used.

2nd EC digit: indicates the bond broken


3rd EC digit: further informaton on group removed

Example:
Code number: EC 4.1.1.22
Systematc name: L-histdine carboxy-lyase

Common name: Histdine decarboxylase


Note the importance of the hyphen and the extra y in the systematc name, because carboxy-lyase and
carboxylase do not mean the same thing: carboxylase simply refers to the involvement of CO2 in a reacton.


Class 5. Isomerases

These enzymes catalyze geometric or structural changes within one molecule. According to the type of
isomerism, they may be called racemases, epimerases, cis-trans-isomerases, isomerases, tautomerases,
mutases or cycloisomerases.

2nd EC digit: indicates type of isomerism
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3rd EC digit: indicates type of substrate


Example:
Code number: EC 5.2.1.10
Systematc name: Maleate cis-trans isomerase
Common name: Maleate isomerase


Class 6. Ligases

Ligases are enzymes catalyzing the joining of two molecules coupled with the hydrolysis of a diphosphate
bond in ATP or a similar triphosphate.

2nd EC digit: indicates the bond formed


3rd EC digit: (only used in the CN ligases)


Example:
Code number: EC 6.3.1.2
Systematc name: L-glutamate: ammonia ligase
Common name: glutamate-ammonia ligase


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Summary:
Class Type of Reacton Catalyzed
Example
(EC number, Common name)
Oxidoreductases
Oxidaton and reducton (removal or
additon of hydrogen atoms)
1.1.1.27
Lactate dehydrogenase
Transferases
Transfer of chemical group (phosphate,
methyl) from one substance to another
2.7.12
Glucokinase
Hydrolases Hydrolysis (splitng of bonds by water)
3.2.1.22
-D-galactosidase
Lyases
Removal of chemical groups from
substances
4.2.1.1
Carbonic anhydrase
Isomerases
Structural rearrangements of atoms
within one molecule
5.3.1.9
Glucose 6-phosphate isomerase
Ligases
Joining together of two molecules
utlizing energy (ATP)
6.2.1.1
Acetyl CoA synthetase

Common Enzymes

1. Amylase - enzyme that catalyzes the hydrolysis of starch into sugars. Present in the saliva of
humans and some other mammals
2. Protease - enzymes that break the long chainlike molecules of proteins into shorter
fragments (peptdes). Present in bacteria and plants but are most abundant in animals.
3. Lipase - enzyme that catalyzes the hydrolysis of fats (lipids). Most lipases act at a specifc
positon on the glycerol backbone of lipid substrate.
4. Pectnase - enzyme that breaks down pectn, a polysaccharide found in plant cell walls.
Pectnase enzymes are commonly used in processes involving the degradaton of plant
materials
5. Cellulase - break down the cellulose molecule into monosaccharides ("simple sugars").
These produced chiefy by fungi, bacteria, and protozoans.

Enzyme Functon
Amylase Breaks down starch into sugar
Protease Breaks down proteins into amino acids
Lipase Breaks down fats into faty acids and glycerol
Pectnase Breaks down pectn into simpler molecules
Cellulase Breaks down cellulose into monosaccharides


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pH of some Enzymes
Pepsin is an enzyme released by the chief cells in the stomach and that degrades food proteins
into peptdes
Trypsin is a serine protease from the PA clan superfamily, found in the digestve system of many
vertebrates, where it hydrolyses proteins.
Maltase is an enzyme that breaks down the disaccharide maltose. Maltase catalyzes the
hydrolysis of maltose to the simple sugar glucose.

Enzyme pH Optmum
Lipase (pancreas) 8
Lipase (stomach) 4 5
Pepsin 1.5 1.6
Trypsin 7.8 8.7
Urease 7
Invertase 4.5
Maltase 6.1 6.8
Amylase (pancreas) 6.7 7
Amylase (malt) 4.6 5.2
Catalase 7

Enzymes involved in Partcular Reacton
1. Papaya
Papain - digestng enzyme, single-chained polypeptde with three disulfde bridges
2. Pineapple
Bromelain - protease enzymes, extract derived from the stems of pineapples, contains
chemicals that might interfere with the growth of tumor cells

Page 11 of 37

Industrial Applications

Detergent Industry

Enzyme used as detergent additves stll represents the largest applicaton of industrial enzymes,
both in terms of volume and value. The major component is proteases, but other and very
diferent hydrolases are introduced to provide various benefts, such as the efcient removal of
specifc stains. The most recent introducton of a new enzyme class into a detergent has been the
additon of a mannanase the result of a joint development between Procter and Gamble and
Novozymes. This enzyme helps remove various food stains containing guar gum, a commonly used
stabilizer and thickening agent in food products.

Enzymes for starch conversion

The enzymatc conversion of starch to high fructose corn syrup is a well-established process and
provides a beautful example of a bioprocess in which the consecutve use of several enzymes is
necessary. The enzymes utlized in the starch industry are also subjected to constant
improvements. The frst step in the process is the conversion of starch to oligomaltodextrins by
the acton of -amylase. The concomitant injecton of steam puts extreme demands on the
thermostability of the enzyme. New -amylases with optmized propertes, such as enhanced
thermal stability, acid tolerance, and ability to functon without the additon of calcium, have
recently been developed ofering obvious benefts to the industry.

Fuel alcohol producton

In the alcohol industry, the use of enzymes for the producton of fermentable sugars from starch
is also well established. Over the past decade, there has been an increasing interest in fuel alcohol
as a result of increased environmental concern, higher crude oil prices and, more acutely, by the
ban in certain regions of the gasoline additve methyl tert-butyl ether (MTBE), which can be
interchanged directly with ethanol. Intense eforts are currently being undertaken to develop
improved enzymes that can enable the utlizaton of cheaper and partally utlized substrates such
as lignocellulose, to make bio-ethanol more compettve with fossil fuels. The cost of enzymes
needed to turn lignocellulose into a suitable fermentaton feed-stock is a major issue, and current
work focuses both on the development of enzymes with increased actvity and stability as well as
on their efcient producton.

Enzymes for the food industry

Applicatons of enzymes in the food industry are many and diverse, ranging from texturizing to
favoring. Common to more or less all food applicatons, the enzymes are applied to processed
food products as processing agents upstream from the fnal product. Several advances have been
Page 12 of 37

made in the optmizaton of enzymes for existng applicatons and in the use of recombinant
protein producton to provide efcient mono-component enzymes that do not have potental
detrimental side-efects. Recently, much work has been carried out on the applicaton of
transglutaminase as a texturing agent in the processing of, for example, sausages, noodles and
yoghurt, where cross-linking of proteins provides improved viscoelastc propertes of the products.
An obstacle, which may prevent even wider usage, is the currently limited availability of the
enzyme in industrial scale. At present only the transglutaminase from Streptovertcillium sp. Is
commercially available at a reasonable scale, and work is ongoing to increase the availability of
the enzyme by recombinant producton in Escherichia coli.

Textle applicatons

In the textle industry a completely new enzymatc actvity has recently been introduced. This
industry is under considerable environmental pressure owing to its large energy and water
consumpton and subsequent environmental polluton. One of the most energy- and water-
consuming steps in the processing of coton is the scouring step, the removal of various remaining
cell-wall components on the cellulose fbers performed at high temperature and under strong
alkaline conditons. An alternatve, enzyme-based process performed at much lower temperatures
and using less water has now been developed based on a pectate lyase. The use of these enzymes
has benefted both the textle industry and the environment.



Page 13 of 37

Factors affecting Enzyme Activity
The activity of an enzyme is affected by its environmental conditions. Changing these alter the
rate of reaction caused by the enzyme. In nature, organisms adjust the conditions of their
enzymes to produce an Optimum rate of reaction, where necessary, or they may have enzymes
which are adapted to function well in extreme conditions where they live.
1. Temperature
Increasing temperature increases the kinetic energy that molecules possess. In a fluid, this means
that there are more random collisions between molecules per unit time.
Since enzymes catalyze reactions by randomly colliding with substrate molecules, increasing
temperature increases the rate of reaction, forming more product.
However, increasing temperature also increases the vibrational energy that molecules have,
specifically in this case enzyme molecules, which puts strain on the bonds that hold them
together.
As temperature increases, more bonds, especially the weaker hydrogen and ionic bonds, will
break as a result of this strain. Breaking bonds within the enzyme will cause the active site to
change shape.
This change in shape means that the active site is less complementary to the shape of the
substrate, so that it is less likely to catalyze the reaction. Eventually, the enzyme will become
denatured and will no longer function.
As temperature increases, more enzymes' molecules' active sites' shapes will be less
complementary to the shape of their substrate, and more enzymes will be denatured. This will
decrease the rate of reaction.
In summary, as temperature increases, initially the rate of reaction will increase, because of
increased Kinetic Energy. However, the effect of bond breaking will become greater and greater,
and the rate of reaction will begin to decrease.













Page 14 of 37

The temperature at which the maximum rate of reaction occurs is called the enzyme's Optimum
Temperature. This is different for different enzymes. Most enzymes in the human body have an
Optimum Temperature of around 37.0 C.
2. pH
H
+
and OH
-
Ions are charged and therefore interfere with hydrogen and ionic bonds that hold
together an enzyme, since they will be attracted or repelled by the charges created by the bonds.
This interference causes a change in shape of the enzyme, and importantly, its active site.
Different enzymes have different optimum pH values. This is the pH value at which the bonds
within them are influenced by H
+
and OH
-
Ions in such a way that the shape of their active site is
the most complementary to the shape of their substrate.
Any change in pH above or below the optimum will quickly cause a decrease in the rate of
reaction, since more of the enzyme molecules will have active sites whose shapes are not (or at
least are less) complementary to the shape of their substrate.


Small changes in pH above or below the optimum do not cause a permanent change to the
enzyme, since the bonds can be reformed. However, extreme changes in pH can cause enzymes
to denature and permanently lose their function.
Enzymes in different locations have different optimum pH values since their environmental
conditions may be different. For example, the enzyme Pepsin functions best at around pH2 and is
found in the stomach, which contains Hydrochloric Acid.

Page 15 of 37

3. Concentration
Changing the enzyme and substrate concentrations affect the rate of reaction of an enzyme-
catalyzed reaction. Controlling these factors in a cell is one way that an organism regulates its
enzyme activity and so its metabolism.
Changing the concentration of a substance only affects the rate of reaction if it is the limiting
factor: that is, it the factor that is stopping a reaction from preceding at a higher rate.
If it is the limiting factor, increasing concentration will increase the rate of reaction up to a point,
after which any increase will not affect the rate of reaction. This is because it will no longer be the
limiting factor and another factor will be limiting the maximum rate of reaction.
As a reaction proceeds, the rate of reaction will decrease, since the substrate will get used up.
The highest rate of reaction, known as the initial reaction rate is the maximum reaction rate for
an enzyme in an experimental situation.


Substrate Concentration
Increasing substrate concentration increases the rate of
reaction. This is because more substrate molecules will be
colliding with enzyme molecules, so more product will be
formed.
However, after a certain concentration, any increase will
have no effect on the rate of reaction, since substrate
concentration will no longer be the limiting factor. The
enzymes will effectively become saturated, and will be
working at their maximum possible rate.


Enzyme Concentration
Increasing Enzyme Concentration will increase the rate of
reaction, as more enzymes will be colliding with substrate
molecules.
However, this too will only have an effect up to a certain
concentration, where the Enzyme Concentration is no
longer the limiting factor.


Page 16 of 37

SIMPLE ENZYME KINETICS

Introducton to Enzyme Kinetcs

Enzyme kinetcs deals with the rate of enzyme reacton and how it is afected by various chemical
and physical conditons. Kinetc studies of enzymatc reactons provide informaton about the
basic mechanism of the enzyme reacton and other parameters that characterize the propertes
of the enzyme. The rate equatons developed from the kinetc studies can be applied in calculatng
reacton tme, yields, and optmum economic conditon, which are important in the design of an
efectve bioreactor.

Important Vocabulary
Chemistry Biochemistry
Reactant Substrate
Catalyst Enzyme


Rate of Reacton

Assume that a substrate (S) is converted to a product (P) with the help of an enzyme (E) in a
reactor as
S
E
P
If you measure the concentratons of substrate and product with respect to tme, the product
concentraton will increase and reach a maximum value, whereas the substrate concentraton will
decrease as shown in the graph.

The rate of reacton can be expressed in terms of either the change of the substrate Cs or the
product concentratons Cp as follows:

Page 17 of 37

In order to understand the efectveness and characteristcs of an enzyme reacton, it is important
to know how the reacton rate is infuenced by reacton conditons such as substrate, product,
and enzyme concentratons. If we measure the inital reacton rate at diferent levels of substrate
and enzyme concentratons, we obtain a series of curves like the one shown in the graph.


From these curves we can conclude the following:

1. The reacton rate is proportonal to the substrate concentraton (that is, frst-order
reacton) when the substrate concentraton is in the low range.

2. The reacton rate does not depend on the substrate concentraton when the substrate
concentraton is high, since the reacton rate changes gradually from frst order to zero
order as the substrate concentraton is increased.

3. The maximum reacton rate rmax is proportonal to the enzyme concentraton within the
range of the enzyme tested.

French chemist Victor Henri (18721940) observed this behavior in 1902 and proposed the rate
equaton

where rmax and KM are kinetc parameters which need to be experimentally determined. This
equaton expresses the three preceding observatons fairly well. The rate is proportonal to Cs
(frst order) for low values of Cs, but with higher values of Cs, the rate becomes constant (zero
order) and equal to rmax. Since this equaton describes the experimental results well, we need to
fnd the kinetc mechanisms which support this equaton.

Page 18 of 37

Formaton of Enzyme-Substrate Complex

Britsh professor Adrian John Brown (1852 1919) proposed that an enzyme forms a complex with
its substrate. The complex then breaks down to the products and regenerates the free enzyme.
The mechanism of one substrate-enzyme reacton can be expressed as

Brown's kinetc inference of the existence of the enzyme-substrate complex was made long
before the chemical nature of enzymes was known, 40 years before the spectrophotometric
detecton of such complexes.

One of the original theories to account for the formaton of the enzyme-substrate complex is the
lock-and-key theory.

Lock-and-Key Theory

In 1894, long before the frst enzyme structure had been determined and several
decades before it had been shown that enzymes are proteins, German chemist
Emil Fischer (18521919) proposed that a substrate molecule fts into the
enzyme actve site like a key in a lock.

An enzyme usually contains one or more actve sites, where reactons with substrates take place.
An actve site may comprise only a few amino acid residues; the rest of the protein is required for
maintaining the three-dimensional integrity of the network. The specifcity of enzymes for
substrates varies from molecule to molecule. Many enzymes exhibit stereochemical specifcity in
that they catalyze the reactons of one conformaton but not the other. Also, enzymes promote
catalysis by positoning key acidic or basic groups and metal ions in the right positon for catalysis.

This theory has been widely embraced, in part because it appears to explain the exquisite
substrate specifcity of enzymes. Just as only the right key will ft into a lock, only the right
substrate fts into the enzyme.

Induced-Fit Model

One limitaton of the Lock and Key Model is that it does not explain why the reacton actually
occurs, and another is that enzymes are fexible and not rigid as this theory implies. The Induced-
Fit Model overcomes some of the limitatons of the Lock and Key Model. In this model, the
substrate stll needs to ft into the enzyme like a key, but instead of simply ftng into the
keyhole, some type of modifcaton is induced in the substrate, enzyme, or both. The
modifcaton begins the process of the reacton.

Page 19 of 37




When the enzyme binds its substrate to form an enzymesubstrate complex, the structure of the
substrate is distorted and pulled into the transiton state conformaton. This reduces the energy
required for the conversion of a given reactant into a product and increases the rate of a reacton
by lowering the energy requirement and therefore increasing the number of efectve collisions
that can result in the formaton of the product.



Products can only be formed when colliding reactants have sufcient actvaton energy. The
greater the actvaton energy for a given reacton is, the lower the number of efectve collisions.

In reality, the free energy diagram for an enzyme-catalyzed reacton is considerably more
complicated than the example. Typically an enzyme-catalyzed reacton will involve multple steps,
each with an actvaton energy that is markedly lower than that for the uncatalyzed reacton.

Michaelis-Menten Kinetc Model
Page 20 of 37


The reacton rate equaton (Michaelis-Menten equaton) can be derived from the preceding
mechanism based on the following assumptons:

1. The total enzyme concentraton stays constant during the reacton, that is

where CE0 is the total enzyme concentraton
CES is the enzyme-substrate complex concentraton
CE is the free-enzyme concentraton

2. The amount of an enzyme is very small compared to the amount of substrate. Therefore,
the formaton of the enzyme-substrate complex does not signifcantly deplete the
substrate.

3. The product concentraton is so low that product inhibiton may be considered negligible.

In additon to the preceding assumptons, there are three diferent approaches to derive the
rate equaton:

1. Michaelis-Menten approach. It is assumed that the product-releasing step is much slower
than the reversible reacton and the slow step determines the rate, while the other is at
equilibrium. This assumpton is known as the Rapid Equilibrium Method. This is an
assumpton which is ofen employed in heterogeneous catalytc reactons in chemical
kinetcs.

2. Briggs-Haldane approach. The change of the intermediate concentraton with respect to
tme is assumed to be negligible, that is

This is also known as the pseudo-steady-state (or quasi-steady-state) assumpton or
simply Steady-state Approximaton in chemical kinetcs and is ofen used in developing
rate expressions in homogeneous catalytc reactons.

3. Numerical soluton. Soluton of the simultaneous diferental equatons developed from
product-releasing step and reversible reacton without simplifcaton. From the
mechanism described by these step and reacton, three rate equatons can be writen for
C, CES and Cs as
Page 21 of 37


These equatons plus the inital assumpton that

can be solved simultaneously without simplifcaton. Since the analytcal soluton of the
preceding simultaneous diferental equatons are not possible, we need to solve them
numerically by using a computer. Among many sofware packages that solve simultaneous
diferental equatons include
Advanced Contnuous Simulaton Language (ACSL, 1975)
Mathematca (Wolfram Research, Inc., Champaign, IL) or
MathCad (MathSof, Inc., Cambridge, MA).



Comparison of the Michaelis-Menten and Briggs-Haldane Approaches


Page 22 of 37

Biographies of Michaelis and Menten

Leonor Michaelis (January 16, 1875 October 8, 1949) was born in Berlin, Germany and graduated from
the humanistc Koellnisches Gymnasium in 1893 afer passing the Abiturienten Examen. It was here that
Michaelis interest in physics and chemistry was frst sparked as he was encouraged by his teachers to
utlize the relatvely unused laboratories at his school.

With concerns about the fnancial stability of a pure scientst, he commenced his study of medicine at
Berlin University in 1893. Among his instructors were Emil du Bois-Reymond for physiology, Emil Fischer
for chemistry, and Oskar Hertwig for histology and embryology.


L. Michaelis (lef) and M. Menten (right)

Maud Menten (March 20, 1879 July 26, 1960) was born in Port Lambton, Ontario and studied medicine
at the University of Toronto (B.A. 1904, M.B. 1907, M.D. 1911). She was among the frst women in Canada
to earn a medical doctorate. She completed her thesis work at University of Chicago. At that tme women
were not allowed to do research in Canada, so she decided to do research in other countries such as the
United States and Germany.

In 1912 she moved to Berlin where she worked with Leonor Michaelis and co-authored their paper in
Biochemische Zeitschrif (1913;49:333369) which showed that the rate of an enzyme-catalyzed reacton
is proportonal to the amount of the enzyme-substrate complex. This relatonship between reacton rate
and enzyme-substrate concentraton is known as the Michaelis-Menten equaton.

Page 23 of 37

Derivaton of Rate Equaton (Michaelis-Menten equaton)

Rapid Equilibrium Method

If the slower reacton

determines the overall rate of reacton, the rate of product formaton and substrate
consumpton is proportonal to the concentraton of the enzyme-substrate complex
as:

From the assumpton that the frst reversible reacton

is in equilibrium, then the forward reacton is equal to the reverse reacton so that
.
Assume that the total enzyme contents are conserved such that
.

Perform substtuton and rearrangement.

This results in the fnal rate equaton

where KM is the Michaelis constant, which characterizes the interacton of an enzyme
with a given substrate; and rmax is the maximum reacton rate, which is proportonal
to the inital enzyme concentraton. (Because of the difculty of expressing the enzyme
concentraton in molar unit, k3 and CEO are lumped into one parameter rmax. Whatever
unit is adopted for CEO, the unit for k3CEO should be the same as r.)

This equaton is known as the Michaelis-Menten equaton and is identcal to the
empirical expression proposed by Henri.


Page 24 of 37

Steady-state Approximaton

From the mechanism

the rates of product formaton and of substrate consumpton are

Assume that the change of CES with tme is negligible compared to that of C or CS.

Substtuton confrms that the rate of product formaton and that of the substrate
consumpton are the same, that is,

Assume that the total enzyme contents are conserved such that

Perform substtuton and rearrangement.

This results in the fnal rate equaton

which is the same as the Michaelis-Menten equaton, except that in the rapid equilibrium
method, KM is equal to the dissociaton constant k2/k1 while in the steady-state
approximaton, it is equal to (k2 + k3)/k1. This can be resolved when k2>>k3, which means
means that the product-releasing step is much slower than the enzyme-substrate complex
dissociaton step.

Since the formaton of the complex involves only weak interactons, it is likely that the rate
of dissociaton of the complex will be rapid. The breakdown of the complex to yield
products will involve the making and breaking of chemical bonds, which is much slower
than the enzyme-substrate complex dissociaton step.

Page 25 of 37

Biographies of Lineweaver and Burk

Hans Lineweaver (December 25, 1907 June 10, 2009) was an American
physical chemist, who developed the LineweaverBurk plot in 1934 while stll a
graduate student, working as a laboratory assistant under Burk at the US
Department of Agriculture in Washington, D.C.

The paper containing the equaton, also known as the Double Reciprocal Plot,
was co-authored by Dr. Dean Burk, and was ttled "The Determinaton of Enzyme
Dissociaton Constants (1934)". It remains the most frequently cited paper ever
to appear in the Journal of the American Chemical Society.



Dean Burk (March 21, 1904 October 6, 1988) was an American biochemist: a
co-discoverer of biotn, medical researcher, and a cancer researcher at the Kaiser
Wilhelm Insttute and the Natonal Cancer Insttute. Burk joined the Department
of Agriculture in 1929 working in the Fixed Nitrogen Research Laboratory. In
1934, he developed the LineweaverBurk plot together with Hans Lineweaver.





Microapparatus used for studying bacterial nitrogen fxaton. Image shows the Warburg-Barcrof microrespiraton apparatus, the Micro-
Kjeldahl distlling apparatus; multple gas-mixing fow meter; nephelometer; and bacterial culture botle.
On the lef is Dr. Burk and to the right is Mr. Lineweaver.
Page 26 of 37

Graphical Analysis

In order to estmate the values of the kinetc parameters, we need to make a series of batch runs
with diferent levels of substrate concentraton. Then the inital reacton rate can be calculated as
a functon of inital substrate concentratons. The results can be ploted graphically so that the
validity of the kinetc model can be tested and the values of the kinetc parameters can be
estmated.

The most straightorward way is to plot r against CS. The asymptote for r will be rmax and is equal
to CS when r = 0.5 rmax. However, this is an unsatsfactory plot in estmatng rmax and because
it is difcult to estmate asymptotes accurately and also difcult to test the validity of the kinetc
model. Therefore, the Michaelis-Menten equaton is usually rearranged so that the results can be
ploted as a straight line.

Lineweaver-Burk plot

Before the inventon of computers, the determinaton of KM and Vmax was a tedious
process. Today curve-ftng programs allow rapid analysis of the data to determine
these values. However, a relatvely simple method allows a relatvely accurate
determinaton of these two constants. This method is to construct a Lineweaver-Burk
plot, also known as a double-reciprocal plot. The basis of a Lineweaver-Burk plot
comes from the manipulaton of the Michaelis-Menten equaton to the form:

This equaton has the form y = mx + b, and describes a straight line. A plot of the
reciprocal of the rate, 1 / V, versus the reciprocal of the substrate concentraton, 1 /
[S], gives a line with a y-intercept equal to 1 / Vmax and an x-intercept of 1 / KM.


Page 27 of 37

Lineweaver-Burk Plot Derivaton

The derivaton starts with the Michaelis-Menten equaton:

=

[]

+[]


Getng the reciprocal of both sides of the equaton, we have:

1

+[]

[]


We can write this equaton as the sum of two fractons over a least common denominator

1

[]
+
[]

[]


which can be simplifed as

[]
+

The derived equaton follows the format for the linear regression equaton

= +

where is equal to
1

is equal to

is equal to
1
[]

is equal to
1

Setng the value of as 0

= + = 0

the x-intercept can be calculated as follows

=

=
1

Page 28 of 37

The Lineweaver-Burk plot is the most widely used graphical technique for the determinaton of
KM and Vmax. However, there are other methods.

Woolf (or Hanes-Woolf or Langmuir) plot

The Woolf plot uses the equaton:

Plotng [S] / V versus [S] gives a straight line.



Eadie-Hofstee plot

An Eadie-Hofstee plot uses the equaton

Plotng V versus V / [S] gives a straight-line.



Page 29 of 37

Woolf Plot Derivaton

The derivaton starts with the Michaelis-Menten equaton:

=

[]

+[]


Getng the reciprocal of both sides of the equaton, we have:

1

+[]

[]


The substrate concentraton value present in the denominator of the right-hand side of the equaton can
be transposed into the lef-hand side of the equaton to give us the value of the rato of the substrate
concentraton to the reacton rate.

[]

+[]

This can be rewriten as

[]

[] +

which follows the format for the linear regression equaton

= +

where is equal to
[]

is equal to
1

is equal to []
is equal to

Setng the value of as 0

= + = 0

the x-intercept can be calculated as follows

=

Page 30 of 37

Eadie-Hofstee Plot Derivaton

The derivaton starts with the Michaelis-Menten equaton:

=

[]

+[]


The sum of the Michaelis constant and the substrate concentraton value present in the denominator of
the right-hand side of the equaton can be transposed into the lef-hand side of the equaton and this
results to:

+[] =

[]

The resultng equaton can be further manipulated as follows.

[] =

[]
=

[]
+

[]
[]


This can be rewriten as

=

[]
+

which follows the format for the linear regression equaton

= +

where is equal to
is equal to

is equal to

[]

is equal to

Setng the value of as 0

= + = 0

the x-intercept can be calculated as follows

=

Page 31 of 37

ENZYME REACTOR WITH SIMPLE KINETICS

A bioreactor is a device within which biochemical transformatons are caused by the acton of enzymes or
living cells. The bioreactor is frequently called a fermenter whether the transformaton is carried out by
living cells or in vivo cellular components. The chemical process in the bioreactor can either be aerobic or
anaerobic. These bioreactors are commonly cylindrical, ranging in size from liters to cubic meters, and are
ofen made of stainless steel.


Industrial and Laboratory Bioreactor

Organisms growing in bioreactors may be submerged in liquid medium or may be atached to the surface
of a solid medium. Submerged cultures may be suspended or immobilized. Suspension bioreactors can
use a wider variety of organisms, since special atachment surfaces are not needed, and can operate at
much larger scale than immobilized cultures. However, in a contnuously operated process the organisms
will be removed from the reactor with the efuent. Immobilizaton is a general term describing a wide
variety of cell or partcle atachment or entrapment. It can be applied to basically all types of biocatalysis
including enzymes, cellular organelles, and animal and plant cells. Immobilizaton is useful for contnuously
operated processes, since the organisms will not be removed with the reactor efuent, but is limited in
scale because the microbes are only present on the surfaces of the vessel.

Page 32 of 37

General Types of Bioreactors

Batch Strred-Tank Reactor

The simplest reactor confguraton for any enzyme reacton is the batch mode. A batch enzyme reactor is
normally equipped with an agitator to mix the reactant, and the pH of the reactant is maintained by
employing either a bufer soluton or a pH controller. An ideal batch reactor is assumed to be well mixed
so that the contents are uniform in compositon at all tmes.


Batch Strred-Tank Reactor

Assume that an enzyme reacton is initated at t = 0 by adding enzyme and the reacton mechanism can
be represented by the Michaelis-Menten equaton:

(1)

An equaton expressing the change of the substrate concentraton with respect to tme can be obtained
by integratng equaton (1), as follows:

(

0
(2)
and

+(

) =

(3)

This equaton shows how CS is changing with respect to tme. With known values of rmax and KM, the
change of CS with tme in a batch reactor can be predicted from this equaton.

Page 33 of 37

Plug-Flow Reactor

In a plug-fow enzyme reactor or tubular-fow enzyme reactor, the substrate enters one end of a cylindrical
tube which is packed with immobilized enzyme and the product stream leaves at the other end. The long
tube and lack of strring device prevents complete mixing of the fuid in the tube. Therefore, the propertes
of the fowing stream will vary in both longitudinal and radial directons. Since the variaton in the radial
directon is small compared to that in the longitudinal directon, it is called a plug-fow reactor. If a plug-
fow reactor is operated at steady state, the propertes will be constant with respect to tme. The ideal
plug-fow enzyme reactor can approximate the long tube, packed-bed, and hollow fber, or multstage
reactor.


Schematc Diagram of a Plug-Flow Reactor

Equaton 3 can also be applied to an ideal steady-state plug-fow reactor, even though the plug-fow
reactor is operated in contnuous mode. However, the tme t in Equaton 3 should be replaced with the
residence tme in the plug-fow reactor.

Rearranging Equaton 3 results in the following useful linear equaton which can be ploted:

ln (

0
/

)
=

ln (

0
/

)
(4)

Page 34 of 37

Contnuous Strred-Tank Reactor

A contnuous strred-tank reactor (CSTR) is an ideal reactor which is based on the assumpton that the
reactor contents are well mixed. Therefore, the concentratons of the various components of the outlet
stream are assumed to be the same as the concentratons of these components in the reactor. Contnuous
operaton of the enzyme reactor can increase the productvity of the reactor signifcantly by eliminatng
the downtme. It is also easy to automate in order to reduce labor costs. Recently, CSTRs are used to
optmize feasible and reliable bioprocess system in order to treat hydrocarbon-rich industrial wastewaters.


Schematc Diagram of a Contnuous Strred-Tank Reactor

The substrate balance of a CSTR can be set up, as follows:
Input - Output + Generaton = Accumulaton

(5)
where F is the fow rate and V is the volume of the reactor contents. It should be noted that rS is the rate
of substrate consumpton for the enzymatc reacton, while dCS /dt is the change of the substrate
concentraton in the reactor. As can be seen in Equaton 5, rS is equal to dCS /dt when F is zero, which is
the case in batch operaton.

For the steady-state CSTR, the substrate concentraton of the reactor should be constant. Therefore, dCS
/dt is equal to zero. If the Michaelis-Menten equaton can be used for the rate of substrate consumpton
(rS), Equaton can be rearranged as:

= =
1

)(

)
(6)
where D is known as diluton rate, and is equal to the reciprocal of the residence tme (). Equaton (6)
can be rearranged to give the linear relatonship:

Michaelis-Menten kinetc parameters can also be estmated by running a series of steady-state CSTR runs
with various fow rates and plotng CS versus (CS)/(CS0-CS).
Summary of the Types of Bioreactors
Page 35 of 37


Category
Batch Stirred-Tank
Reactor
Plug-Flow Reactor
Continuously Stirred-
Tank Reactor
Operation
assumed to be well
mixed; contents are
uniform in composition
at all times
equipped with an
agitator to mix the
reactant; buffer
solution or pH
controller for pH

the substrate enters
one end of a
cylindrical tube which
is packed with
immobilized enzyme
and the product
stream leaves at the
other end
The long tube and lack
of stirring device
prevents complete
mixing of the fluid in
the tube
based on the
assumption that the
reactor contents are
well mixed
the concentrations of
the various components
of the outlet stream are
assumed to be the same
as the concentrations of
these components in
the reactor
Application Composting Waste Treatment
Industrial Wastewater
Treatment
Equations

0
=

+(

)
=

ln (

0
/

)
=

ln (

0
/

= =
1

)(

ENZYME INHIBITION
Inhibitor
- is a modulator (substance which can combine with enzymes to alter their catalytic activities) which decreases enzyme activity. It can decrease
the rate of reaction either competitively, noncompetitively / partially competitively, or uncompetitively.

TYPE OF
INHIBITION
Characteristics Reaction Scheme
Modification to
Michaelis-Menten Eq.
Consequences
Competitive
- competitive inhibitor has a strong
structural resemblance to the substrate
- both the inhibitor and substrate
compete for the active site of an
enzyme
- inhibitor only binds to the free enzyme


KI = [E][I] / [EI]

- Vmax is unchanged
- KM is increased

Non-
Competitive
- inhibitor binds to both E and ES
- Inhibitor binds close to the active site, or
by binding elsewhere on E has an
influence on the active site



- Vmax is decreased
- KM is increased

Uncompetitive
- inhibitor binds directly to the ES
complex
- inhibitor does not have to bind at the
active site
- inhibitor does not have to resemble the
substrate


- Vmax is decreased
- KM is unchanged

Plots based on Lineweaver-Burk:

Figure 1. Competitive Inhibition Figure 2. Non-competitive Inhibition Figure 3. Uncompetitive Inhibition

Bibliography
Bailey, J., & Ollis, D. (1986). Biochemical Engineering Fundamentals. New York: McGraw-Hill Book Co.
Chang, R. (2005). Physical Chemistry for the Biosciences. California: University Science Books.
Dutta, R. (2008). Fundamentals of Biochemical Engineering. New Delhi, India: Ane Books India.
Marangoni, A. G. (2003). Enzyme Kinetics: A Modern Approach. New Jersey: John Wiley & Sons, Inc.
Moore, J. T., & Langley, R. (New Jersey). Biochemistry For Dummies. 2008: Wiley Publishing, Inc.
Pratt, C., & Cornely, K. (2014). Essential Biochemistry (3rd ed.). New Jersey: John Wiley & Sons, Inc.
Rogers, A., & Gibon, Y. (2009). Enzyme Kinetics: Theory and Practice. In J. Schwender (Ed.), Plant
Metabolic Networks (pp. 71-103). Springer Science+Business Media, LLC.