Ms Banda (BPharm), MSc Biochem

1
OBJECTIVES
 List the properties of enzymes

 Classify enzymes and explain the type of reactions

catalysed

 Explain the catalytic action, enzyme kinetics and

clinical importance of enzymes

2
 Introduction to Enzymes
 Enzymes are biological polymers that catalyze the chemical reactions
which make life as we know possible.

 An enzyme is a biological catalyst produced by biological cells which

speeds up the rate of a reaction

 Enzymes are produced by living tissue

 They increase the rate of a reaction but DO NOT change the reaction

 Substances on which enzymes act to convert them into products are

called substrates.

 For chemical reaction to take place, the reacting molecules are required
to gain a minimum amount of energy, called energy of activation.

1/15/2019 3
  Enzymes have immense catalytic powers and accelerate reactions at
least a million times by reducing the energy of activation.

 Few enzymes are simple proteins while some are conjugated proteins.

 In such enzymes the non-protein part is called prosthetic group or

coenzyme and the protein part is called apoenzyme.

 When many different enzyme catalyzing sites are located at different

sites of the same macromolecule, it is called multienzyme complex.
Examples: fatty acid synthase, carbamoyl phospahte synthetase II,
pyruvate dehydrogenase, etc.

 The complex becomes inactive when it is fractionated into smaller

units each bearing individual enzyme activity.

1/15/2019 4
 BIOMEDICAL IMPORTANCE
 The presence and maintenance of a complete and balanced set of
enzymes is essential for ;

- the breakdown of nutrients to supply energy and chemical building

blocks;

- the assembly of those building blocks into proteins, DNA,

membranes, cells, and tissues; and

- the harnessing of energy to power cell motility and muscle

contraction.

 Deficiencies in the quantity or catalytic activity of key enzymes can

result from genetic defects, nutritional deficits, or toxins.

1/15/2019 5
 Properties of Enzymes
1. Enzymes speed up the rate of a reaction by a factor of at least 106

2. Enzymes are neither consumed no permanently altered as a

consequence of their participation in a reaction

3. Enzymes are specific for both the type of reaction catalysed and for a
single substrate or a small set of closely related substrates

4. Nearly all enzymes are proteins. The protein enzymes can get
denatured by extreme heat, strong acids or bases and work in a
narrow range of pH and temperature

1/15/2019 6
 Properties of enzymes cont’d
5. Are stereospecific and only catalyse reactions only of specific
stereoisomers e.g. D- but no L-sugars

6. Enzymes require co-factors/co-enzymes e.g. Biotin, NAD+,

FMN, FAD

7. They transform one form of energy into a more usable form

1/15/2019 7
 Co-enzymes and Co-factors
CO-ENZYMES CO-FACTORS

 Prosthetic groups are  Bind in a transient

distinguished by their tight, dissociable manner either to
stable incorporation into a the enzyme or to a substrate
protein structure by either
covalent or non covalent  Must be present in the
forces surrounding medium of the
enzyme for catalysis to occur
 Examples include
tertrahydrofolate, NAD+,  Enzymes requiring a metal
NADP, FMN, FAD, Co, Cu, Zn ion co-factor are termed
etc. metal activated enzymes
 Enzymes containing tightly
bound metal ions are termed
metalloenzymes
1/15/2019 8
 Metal ions
 Metal ions in redox reactions generally are complexed to prosthetic
groups such as heme or ion sulphur clusters

 They may facilitate binding or orientation of substrates, formation of

bonds with reaction intermediates, or interaction with substrates to
render them more electrophilic (electron-poor) or nucleophilic
(electron-rich)

 Note: nearly a third of all known enzymes require one or more metal
ions for catalysis

1/15/2019 9
 NOMENCLATURE AND CLASSIFICATION OF ENZYMES

 The International Union of Biochemistry (IUB) adopted a

nomenclature system based on chemical reaction type and reaction
mechanism.

 The systemic names for enzymes include the substrate and the type of
reaction.

 According to this system, enzymes are grouped into six main classes.
They are:

1/15/2019 10
 No Class Type of reaction catalyzed
1 Oxidoreductases Catalyse oxidation reduction reaction of their substrates
e.g. alcohol dehydrogenase and lactate dehydrogenase

2 Transferases Catalyse the transfer of a particular group from one

substrate to another e.g. hexokinase

3 Hydrolases Catalyse the hydrolytic cleavage (addition or removal of

water)to C-C, C-O, C-N, P-O and other certain bonds
including acid anhydride bonds e.g. glucose 6
phosphatase

4 Lyases Enzymes catalyzing addition of groups to double bonds,

or formation of double bonds by removal of groups e.g.
fumarase

5 Isomerases Enzymes catalyzing transfer of groups within molecules

to yield isomeric forms e.g. epimerase

6 Ligases Enzymes catalyzing formation of C-C, C-S, C-O, and C-N

bonds by condensation reactions coupled to ATP
hydrolysis

1/15/2019 11
 SPECIFICITY OF ENZYMES
 An important property of enzyme is their specificity. Specificity is of 3
different types;

1. Optical specificity:
 Substrates can have optical isomers, but only one of the isomers acts as
a substrate for the enzyme activity.

2. Reaction specificity:
 An enzyme can catalyze only a single type of reaction.
 A substrate can undergo many reaction, each reaction catalysed by
different enzymes.

1/15/2019 12
 3. Substrate specificity:
 This means that certain enzymes are specific for a certain substrate.
 Substrate specificity is of two type; group dependent and bond
dependent.

 Group specificity - the enzyme will act only on molecules that have
specific functional groups, such as amino, phosphate and methyl
groups.
- E.g. Trypsin hydrolyses the residues of only lysine and arginine,
chymotrypsin hydrolyses residues of only aromatic amino acids.

 Bond specificity - the enzyme will act on a particular type of chemical

bond regardless of the rest of the molecular structure.
- E.g. Proteolytic enzymes, glycosidases and lipases act on peptide,
glycosidic and ester bonds respectively.
1/15/2019 13
MECHANISM OF ENZYME ACTION
 According to most acceptable hypothesis, enzyme molecule (E) first
combines with substrate molecule (S) to form an enzyme-substrate (ES)
complex which further dissociates to form product (P) and enzyme (E).

 Enzyme once dissociated from ES complex is free to combine with another

substrate and form product.
 The ES complex is an intermediate or transient complex held together by weak
non-covalent bonds such as H-bonds, Van der Waals forces, hydrophobic
interactions.
 The site at which the substrate can bind to the enzyme with extreme specificity
is called active site or catalytic site.
 The active site is made up of several amino acids that come together as a result
of folding of secondary and tertiary structures of the enzyme.

1/15/2019 14
 MODELS OF ENZYME-SUBSTRATE COMPLEX FORMATION
1) Template or Lock and Key Model
 This model states that the active site already exists in proper
conformation even in the absence of the substrate.
 The active site provides a rigid, pre-shaped template fitting with the
size and shape of the substrate molecule.
 Substrate fits into the active site as key fits into lock, hence called lock
and key model.
 Model cannot explain change in enzyme activity in presence of
allosteric modulators.

2) Induced Fit or Koshland Model

 Important feature of this model is the flexibility of active site region.
 According to this, the substrate during its binding induces
conformational changes in the active site to attain the final catalytic
shape and form.
 The change is analogous to placing a hand (substitute) into a glove
(enzyme)
1/15/2019 15
  This model explains;
- enzymes become inactive on denaturation
- saturation kinetic
- competitive inhibition
- allosteric modulation

1/15/2019 16
ENZYMES EMPLOY MULTIPLE MECHANISMS TO FACILITATE
CATALYSIS
Four general mechanisms:

Catalysis by Proximity
 For molecules to react, they must come within bond forming distance
of one another.

 The higher their concentration, the more frequently they will

encounter one another and the greater will be the rate of their reaction.

 When an enzyme binds substrate molecules in its active site, it orients

the substrate molecules spatially in a position ideal for them to
interact.

1/15/2019 17
 Acid-Base Catalysis
 Most cellular reactions require positive and negative charges within the
molecules to allow reaction.

 The enzyme can add or remove protons from the substrate, changing
its charge; or from a neutral group of the enzyme becomes ionic.

 The removal of a proton makes a group more nucleophilic, which is a

group (nucleophile) that will attack a positive centre.

 Similarly, enzymes can protonate a group making them positive and

more prone to nucleophilic attack.

 The addition and removal of protons is referred to as general acid and

general base catalysis, respectively.

1/15/2019 18
  The advantage of using general acid–base catalysis is that at
physiological neutral pH, enzymes can catalyze a reaction even though
the concentration of OH- and H+ is low.

Catalysis by Strain
 Enzymes that catalyze lytic reactions typically bind their substrates in a
conformation slightly unfavorable for the bond that will undergo
cleavage.

 The resulting strain stretches or distorts the targeted bond, weakening

it and making it more vulnerable to cleavage.

1/15/2019 19
 Covalent Catalysis
 This catalysis involves the formation of a covalent bond between the
enzyme and one or more substrates.

 The modified enzyme then becomes a reactant.

 The chemical modification of the enzyme is, however, transient.

 Covalent catalysis is particularly common among enzymes that

catalyze group transfer reactions.

1/15/2019 20
The Active Sites of Enzymes Have Some Common Features

1. The active site is a three-dimensional cleft formed by amino acid

groups that are distant from the active site.

2. The active site takes up a relatively small part of the total volume of an
enzyme.

3. Substrates are bound to enzymes by multiple weak attractions.

4. Most of the amino acids serve as a large scaffold to allow for the proper
alignment of the functional groups of the substrate.

5. The specificity of binding depends on the precisely defined

arrangement of atoms in an active site.

1/15/2019 21
FACTORS AFFECTING ENZYMES
 Temperature

 pH

 Enzyme concentration

 Substrate concentartion

 Inhibitors

1/15/2019 22
 1. Temperature
 As the temperature rises, reacting molecules gain kinetic energy and
this increases the chances of a successful collision and so the rate
increases.

 Each enzyme is most active at a specific temperature, called optimum

temperature, which in humans is the body temperature (37.5 oC)

 Above this temperature the enzyme denatures

since at higher temperatures intra- and
intermolecular bonds are broken as the
enzyme molecules gain even more kinetic
energy.

 The Q10 or temperature coefficient is a

measure of the rate of change of a
biological or chemical system as a
consequence of increasing the
temperature by 10 °C.
1/15/2019 23
 2. pH
 The pH at which an enzyme exhibits greatest activity is called optimal
pH.
 Small changes in pH above or below the optimum do not cause a
permanent change to the enzyme, since the bonds can be reformed.
 Extreme changes in pH causes distortion of the active site such that
substrates no longer fit and the enzyme is said to have denatured
 H+ and OH- Ions are charged and therefore interfere with Hydrogen
and Ionic bonds that hold together an enzyme i.e. polar side chain
groups begin to repel each other or attract each other when at optimal
pH they did not.
 This interference causes a
change in shape of the
enzyme, and importantly,
its active site.

1/15/2019 24
 3. Enzyme Concentration
 Rate of enzyme activity is directly proportional to
enzyme concentration as long as the substrate
concentration is in excess.

4. 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.
 After a certain concentration, any increase will have no
effect on the rate of reaction, because enzymes will
effectively become saturated.
 The enzyme-substrate complex has to dissociate before
the active sites are free to accommodate more substrate.

1/15/2019 25
 5. Inhibitors

 Enzyme inhibitors are substances which alter the catalytic action of the enzyme
and consequently slow down, or in some cases, stop catalysis.

 Whenever the active site is not available for binding of the substrate the
enzyme activity may be reduced.

1/15/2019 26
 ENZYME INHIBITION
 The chemical substances which inactivate enzymes are called inhibitors and
the process is called enzyme inhibition.

 Enzymes catalyze virtually all cellular processes, enzyme inhibitors are among
the most important pharmaceutical agents known.

 For example, aspirin (acetylsalicylate) inhibits the enzyme that catalyzes the
first step in the synthesis of prostaglandins, compounds involved in many
processes, including some that produce pain.

 Three major groups of inhibition:

1. Reversible inhibition
2. Irreversible inhibition
3. Allosteric inhibition

1/15/2019 27
 Reversible inhibition.

 When the active site or catalytic site is occupied by a substance other than the
substrate, its activity is inhibited.

 One common type of reversible inhibition is called competitive inhibition.

 A competitive inhibitor [I] competes with the substrate for binding the active
site of a free enzyme.

 While the inhibitor occupies the active site it prevents binding of the substrate
to the enzyme.

 Many competitive inhibitors are compounds that resemble the substrate and
combine with the enzyme to form an EI complex, but without leading to
catalysis.

1/15/2019 28
  Because the inhibitor binds reversibly to the enzyme, the inhibition can be
overcome by adding more substrate.

Clinical Significance:
 Medical therapy based on competitive inhibition is used to treat patients who
have ingested methanol, a solvent found in gas-line antifreeze.

 The liver enzyme alcohol dehydrogenase converts methanol to formaldehyde,

which is damaging to many tissues especially eyes.

 Ethanol competes effectively with methanol as an alternative substrate for

alcohol dehydrogenase converting it to acetaldehyde.

 This slows the formation of formaldehyde, lessening the danger while the
kidneys filter out the methanol to be excreted harmlessly in the urine.

1/15/2019 29
  Allopurinol is a drug used to treat gout. Uric acid is formed in the body by
oxidation of hypoxanthine by the enzyme xanthine oxidase. Allopurinol acts as
a competitive inhibitor of xanthine oxidase reducing uric acid formation.

 Methotrexate is used in cancer therapy. It’s a structural analog of folic acid. It

inhibits folate reductase and prevents formation of FH4, which in turn inhibits
DNA synthesis.

 Succinylcholine is used as a muscle relaxant. It is structurally similar to

acetylcholine. It competitively binds to post-synaptic receptors.
Acetylcholinesterase cannot hydrolyse them which causes continued
depolarization resulting in muscle relaxation.

 Dicoumarol is used as an anticoagulant, structurally similar to vitamin K and

competitively inhibits vitamin K epoxide reductase, an enzyme that
recycles vitamin K.

1/15/2019 30
  An noncompetitive inhibitor binds reversibly to both the free enzyme and
the ES complex.

 Non-competitive inhibition is observed only for enzymes with two or more

substrates.

 The enzymes have a permanent allosteric site that can bind inhibitors

 These non competitive inhibitors do not compete for the active site with the
substrate

 Non competitive inhibitors bind to the enzyme regardless of whether the

substrate is bound or not

 These inhibitors bring about changes in the 3D structure of the enzyme

thereby inactivating it.

1/15/2019 31
  Uncompetitive inhibitor s are reversible inhibitors that can bind
to the ES complex rather than the free enzyme, binding to an
alternative binding site.

 In contrast to competitive inhibition, increasing the concentration of

substrate will not overcome the effect of the inhibitor because [I] binds
to ES complex and not the free enzyme.

 The inhibitor only binds after the enzyme substrate complex is formed
because only upon formation of this complex will the enzyme form a
pocket upon which the inhibitor can bind

1/15/2019 32
 Irreversible inhibition

 The irreversible inhibitors are those that bind covalently (or non
covalently)with or destroy a functional group on an.

 Irreversible inhibitors can bind so tightly to the enzyme such that they
dissociate very slowly and therefore seem irreversible

 Other irreversible inhibitors can also chemically modify and inactivate the
enzymes.

 If an irreversible inhibitor can be removed only at the loss of enzyme activity, it

is known as irreversible non-competitive inhibition.

 Examples of irreversible inhibitors include aspirin and penicillin which bind to

cyclo-oxygenase and transpeptidase respectively

1/15/2019 33
 Clinical Significance
 British anti Lewesite (BAL) is used as antidote for heavy metal poisoning.
Heavy metals inhibit enzymes by reacting with –SH groups. BAL provides –SH
for the heavy metals to act on.

 Disulfuram used in treatment of alcoholism. It inhibits aldehyde

dehydrogenase preventing oxidation of acetaldehyde which accumulates
producing sickening effect leading to aversion to alcohol.

 Suicide inhibition is a special type of irreversible non-competitive inhibition

in which the substrate analog is converted to a more effective inhibitor with
the help of the enzyme to be inhibited.

 The new inhibitor formed binds irreversibly with the enzyme.

 Examples include allopurinol, aspirin, 5-fluorouracil.

1/15/2019 34
 Allosteric inhibition

 It is a kind of inhibition when the inhibitor binds to the enzyme at a

site other than the active site, sometimes on a different region in the
enzyme called allosteric site.

1/15/2019 35
 REGULATION OF ENZYME ACTIVITY
 Regulatory enzymes exhibit increased or decreased catalytic activity in
response to certain signals.

 These enzymes allow the cell to meet changing needs for energy and for
biomolecules required in growth and repair.

 Allosteric enzymes function through reversible, non-covalent binding of

regulatory compounds called allosteric modulators or allosteric effectors,
which are generally small metabolites or cofactors.

 Changes in enzyme-substrate interaction due to the allosteric effects of

regulatory molecules other than the substrate are called heterotropic alloteric
modulation.

 When the binding of substrate enhances the interaction between the allosteric
enzyme and more substrate molecules is called homotropic allosteric effects.

1/15/2019 36
  Binding of the allosteric effector brings about conformational changes of the
enzyme so the affinity for the substrate or other ligands also changes.

 Positive (+) allosteric effectors increase the enzyme affinity for the substrate.
The reverse is true for negative (-) effectors.

 Allosteric site at which the positive effector binds is called activator site,
negative effector binds at an inhibitory site.

 Feedback regulation is when the product inhibits or activates the enzyme

activity in response to stimuli.

 If the end product becomes available in the environment, it is unnecessary and

wasteful for the cells to continue to produce the product. Cells have the ability
to shut down a pathway when it is not needed.

1/15/2019 37
  In biochemical pathways, the product of one reaction becomes the substrate
for the next reaction.

 When the regulatory enzyme reaction is slowed, all subsequent enzymes

operate at reduced rates as their substrates are depleted.

 After the product has been utilized and its concentration decreased, the
inhibition is relaxed, and the formation of the product resumes.

 Example: Dietary cholesterol restricts the synthesis of cholesterol from acetate

in mammalian tissues.

1/15/2019 38
  Other enzymes are regulated by reversible covalent modification.

 These include the phosphorylation, adenylation, acetylation, uridylation, ADP-

ribosylation, and methylation of enzymes.

 The covalently attached groups are removed from the enzyme by separate
enzymes.

 Phosphorylation is the most common type of regulatory modification found in

eukaryotes. It is the addition of phosphate group by protein kinases to serine,
threonine, or tyrosine residues on specific enzymes.

 An important example of regulation by phosphorylation is observed in the

enzyme glycogen phosphorylase of muscle and liver.

1/15/2019 39
 Coenzymes
Coenzymes ( complex organic or metalloorganic molecules) serve as
substrate shuttles
 They transport substrates from their point of generation to their point of
utilization
Examples:
 methyl groups (folates),
 acyl groups (coenzyme A),
 oligosaccharides (dolichol)

 A complete, catalytically active enzyme together with its bound coenzyme

and/or metal ions is called a holoenzyme
 The protein part of an enzyme is called the apoenzyme or opoprotein

40
Derivatives of B vitamins
Many coenzymes, cofactors and prosthetic groups are derivatives of B vitamins-Water-
soluble B vitamins supply important components of numerous coenzymes
Coenzyme Examples of chemical Dietary precursor in mammals
groups transferred
Biocytin CO2 Biotin
Coenzyme A Acyl groups Pantothenic acid
5’-deoxyadenosyl H atoms and alky Vitamin B12
cobalamin groups
Flavin adenine Electrons Riboflavin (Vitamin B2)
dinucleotide (FAD)
Lipoate Electrons and Not required in diet
acyl groups
Nicotinamide adenine Hydride ion (:H─) Nicotinic acid (Niacin)
dinucleotide (NAD)
Pyridoxal phosphate Amino acids Pyridoxine (Vitamin B6)
Tetrahydrofolate One-carbon groups Folate
Thiamine pyrophosphate Aldehydes Thiamine (Vitamin B1)

-many in addition contain the adenine, ribose, and phosphoryl moieties of AMP or ADP e.g.
NAD, NADP, FAD,

41
Enzymes in clinical practice
The analysis of certain enzymes aids in diagnosis

 Enzymes that fulfil functions indispensable to cell viability are present

through out body tissues

 Other enzymes or isoenzymes are expressed only in specific tissues,

during certain periods of development, or in response to specific
physiological and pathophysiological changes

 Note: Analysis of the presence and distribution of enzymes and

isoenzymes- whose expression is normally tissue-, time- or
circumstances-specific often aids diagnosis

42
Enzymes in clinical practice cont’d
Nonfunctional plasma enzymes aid diagnosis and prognosis

 Certain enzymes, proenzymes, and their substrates are present at all

times in the circulation of normal individuals and perform physiologic
functions in blood

 These are also known as functional plasma enzymes .

 lipoprotein lipase, pseudo cholinesterase, proenzymes of blood
coagulation and blood clot dissolution
 Majority are synthesized and secreted by the liver

43
Enzymes in clinical practice cont’d
 Plasma also contain other enzymes of no known physiological function
in blood

 These are known as nonfunctional plasma enzymes

 Arise from normal destruction of erythrocytes, leukocytes, and
other tissues
 tissue damage or disease is generally accompanied by increase in
the levels of several nonfunctional plasma enzymes

44
Serum Enzyme Major diagnostic use

Amino transferases
Aspartate amino transferase (SGOT) Myocardial infarction
Alanine amino transferase (SGPT) Viral hepatitis

Amylase Acute pancreatitis

Ceruloplasmin Hepatolenticular degeneration

(Wilson’s disease)
Creatine kinase Muscle disorders and myocardial
infarction
γ-Glutamyl transpeptidase Various viral diseases

Lactate dehydrogenase Myocardial infarction

(isoenzymes)
Lipase Acute pancreatitis
Phosphatase, acid Metastatic carcinoma of the
prostate
Phosphatase, alkaline Various bone disorders, obstructive
(isoenzyme) liver disease

45
 KINETIC PROPERTIES OF ENZYMES
 In enzyme kinetics, the reaction rate is measured and the effects of varying the
conditions of the reaction are investigated.

 One of the first things that is measured in kinetics is the variation in rate of
reaction with substrate concentration.

 The rate of catalysis rises linearly as substrate concentration increases and then
begins to level off and approach a maximum at higher substrate concentrations.
 The saturation effect shows that all the enzyme binding sites are occupied by
substrates.

 Thus the reaction velocity becomes independent of substrate concentration.

 The simplest model which accounts for this behavior is:

------ (1)

 k1 the rate constant of the forward reaction of E+S, k-1 the rate constant of the
reverse reaction where the ES dissociates to E+S and k2 the rate constant of the
forward reaction of ES forming E+P.
 The initial velocity, V0 is determined by the rate of dissociation of [ES] whose
rate constant is k2,
V0 = k2 [ES] ------ (2)

 Since ES is an intermediate and its concentration unknown, we have to express

[ES] in terms of known values.

Rate of formation of [ES] = k1 [E] [S] ------ (3)

Rate of dissociation of [ES] = (k-1 + k2) [ES] ------ (4)

 The steady state occurs when rate of formation of ES is equal to rate of

dissociation of ES.
k1 [E] [S] = (k-1 + k2) [ES] ------ (5)
 Rearranging,
[E] [S] = k−1 + k2 ------ (6)
[ES] k1

 This can be simplified by defining a new constant, KM, called the Michaelis
constant:
KM = k−1 + k2 ------ (7)
k1
 Substituting (7) into (6),
[ES] = [E] [S] ------ (8)
KM

 Since in most situations the enzyme concentration is very small ([E] << [S]),
the concentration of the uncombined S is almost equal to the total
concentration of S.
 The concentration of uncombined enzyme [E] is equal to the total enzyme
concentration [ET] minus the concentration of the ES complex.
[E] = [ET] – [ES] ------ (9)

 Substituting this expression for [E] in (8) gives,

[ES] = ([ET]− [ES])[S] ------ (10)
KM
 Substituting (7) in (10),
[ES] = ([ET]− [ES])[S] ------ (11)
k−1 + k2 / k1

 A series of algebraic steps follows,

k1 ([ET] – [ES]) [S] = k−1 [ES] + k2 [ES] ------ (12)

k1 [ET][S] - k1 [ES][S] = (k−1 + k2) [ES] ------ (13)

 Adding the term k1[ES][S] to both sides of the equation and simplifying gives
k1[ET][S] = (k1[S] + k-1 + k2)[ES] ------ (14)

 We then solve this equation for [ES]:

[ES] = k1[ET][S] ------ (15)
k1[S] + k-1 + k2

 This can now be simplified further, combining the rate constants into one
expression:
[ES] = [ET][S] ------ (16)
[S] + (k-1 + k2) / k1
 Substituting KM,
[ES] = [ET][S] ------ (17)
KM + [S]
 We can now express V0 in terms of [ES],
V0 = k2 [ET][S] ------ (18)
KM + [S]

 This equation can be further simplified. Because the maximum velocity occurs
when the enzyme is saturated (that is, with [ES] = [ET]). Vmax can be defined as
k2[ET].
 Substituting this in (18) gives,
V0 = Vmax [S] ------ (19)
KM + [S]

 This is the Michaelis-Menten equation, the rate equation for a one-substrate

enzyme-catalyzed reaction.
 When V0 is one half of Vmax then,
Vmax = Vmax [S]
2 KM + [S]
 ½ = [S] / KM + [S]

KM + [S] = 2 [S]
Or ,
KM = [S] ; when V0 = ½ Vmax
 END

54