Enzymology basics

1. Basic enzymology

2. Early Enzyme Discoveries
• The existence of enzymes has been known for well over a
century. Some of the earliest studies were performed in
1835 by the Swedish chemist Jon Jakob Berzelius who
termed their chemical action catalytic. It was not until 1926,
however, that the first enzyme was obtained in pure form, a
feat accomplished by James B. Sumner of Cornell University.
Sumner was able to isolate and crystallize the
enzyme urease from the jack bean. His work was to earn
him the 1947 Nobel Prize.
• John H. Northrop and Wendell M. Stanley of the Rockefeller
Institute for Medical Research shared the 1947 Nobel Prize
with Sumner. They discovered a complex procedure for
isolating pepsin. This precipitation technique devised by
Northrop and Stanley has been used to crystallize several
enzymes.

3. What is an Enzyme?
• Enzymes
- Are the most remarkable and highly specialized protein
- Catalyze hundreds of stepwise reactions in biological systems
- Regulate many different metabolic activities necessary to sustain life
In short,
Living systems use catalyst called enzymes to increase the rate of
chemical reactions

4. Why to study enzymology
The study of enzymes has immense practical importance:
- In medical science: to know the epidemiology, to diagnose, and to treat
diseases ( inheritable genetic disorders)
- In chemical industries
- In food Processing
- In agriculture
- In everyday activities in the home ( food preparation, cleaning, beauty
care etc.)

5. Chemical Nature of Enzymes
All known enzymes are proteins. They are high molecular weight compounds made
up principally of chains of amino acids linked together by peptide bonds. See Figure
1.
Enzymes can be denatured and precipitated with salts, solvents and
other reagents. They have molecular weights ranging from 10,000 to
2,000,000.

6. Enzymes vs. Nonbiological catalysts
• Enzymes have their reaction specificity
• The activities of many enzymes are regulated so that the
organism can respond to the changing condition or follow
genetically determined developmental programs
• Enzyme activity is affected by the temperature and pH of
the medium

7. Properties of enzymes…
• Some enzymes require no chemical group other than their amino acid
residue for activity.
• Some enzymes may require an additional chemical compound called
cofactors like metal ion activators (Zn2+, Mg2+, Fe2+, Mn2+etc.)
• Some enzymes may require a complex orgainc or metelloorganic molecule
called coenzymes. A coenzyme is a non-protein organic substance which is
dialyzable, thermostable and loosely attached to the protein part.
• Sometimes they need a prosthetic group - an organic substance which is
dialyzable and thermostable which is firmly attached to the protein or
apoenzyme portion.
•
• Apoenzyme + Cofactor = Holoenzyme

8. How do enzymes work ?
• An enzyme circumvents many problems providing a
specific environment within which a given reaction is
energetically more favorable.
• An enzyme-catalyzed reaction occurs within the
confinement of a pocket on the enzymes called the active
site
• The molecule that is bound by the active site and acted
upon by the enzyme is called the substrate
• The enzyme-substrate complex is central to the action of
enzymes.

9. Enzyme specificity
Enzymes are specific and efficient catalyst
In nonenzymatic reactions, substrates are transformed into product by bond-
making and bond-breaking processes. In an enzymatic reaction, these
processes occur through interactions of the substrates with appropriate
side chains of the amino acid residues of the enzyme or with the coenzyme
of the enzyme.
- One property of many enzymes is specificity: catalyzing one
chemical reaction with only one substrate
An Example is Carbonic anhydrase. This enzyme catalyzes only one reversible
reaction, the breakdown of carbonic acid (H2CO3) to water and carbon
dioxide. At ideal conditions, a single molecule of Carbonic anhydrase is
capable of catalyzing the breakdown of about 36 million molecules of
carbonic acid in one minute.

10. A few enzymes exhibit absolute specificity; that is, they will catalyze only
one particular reaction. Other enzymes will be specific for a particular type
of chemical bond or functional group. In general, there are four distinct
types of specificity:
Absolute specificity - the enzyme will catalyze only one reaction.
Group specificity - the enzyme will act only on molecules that have specific
functional groups, such as amino, phosphate and methyl groups.
Linkage specificity - the enzyme will act on a particular type of chemical
bond regardless of the rest of the molecular structure.
Stereochemical specificity - the enzyme will act on a particular steric or
optical isomer.
Though enzymes exhibit great degrees of specificity, cofactors may serve
many apoenzymes. For example, nicotinamide adenine dinucleotide (NAD)
is a coenzyme for a great number of dehydrogenase reactions in which it
acts as a hydrogen acceptor. Among them are the alcohol
dehydrogenase, malate dehydrogenase and lactate
dehydrogenase reactions.

11. How do enzymes work?
• In a biological reaction, the reacting species must come together and
undergo electronic rearrangement that result in the formation of a
product. Let us consider an idealized transfer reaction in which
compound A—B reacts with compound C to form two new products, A
and B—C.
• In order for the first two compounds to react, they must approach
closely enough for their constituent atoms to interact.
• Normally, atoms that approach too closely repel each other; but if the
groups have sufficient free energy, they can pass this point and react
with each other to form products.
• The energy requiring step of the reaction is known as an energy barrier,
called as free energy of activation or activation energy (G‡)

12. Idealized transfer reaction

13. Reaction coordinate diagram for the reaction A—B + C A + B—C
.
The progress of the
reaction(reaction
coordinate) is shown on
the horizontal axis, and the
free energy(G) is shown on
the vertical axis. The
transition state of the
reaction, represented as
a..b..c, is the point of
highest free energy.The
free energy difference
between the reactant and
the transition state is the
free energy of activation
(G‡).

14. Reaction coordinate diagram of a reaction:
Here reactants and products have different free energies
When the free energy of the reactant is greater than that of the product, the free
energy changes for the reaction is negative, so the reaction proceeds
spontaneously (a). When the free energy of the product is greater than that of the
reactant, the free energy change of the reaction is positive, so the reaction does
not proceed spontaneously (however the reverse reaction does proceed).

15. Effect of a catalyst on a chemical reaction
Here the reactants
proceed through a
transition state (X‡) during
their conversion to
products. A catalyst lowers
the free energy of
activation ( G‡) for the
reaction so that  G‡
cat <
G‡
uncat. Loweing the free
energy of the transition
state (X‡) accelerates the
reaction because more
reactants are able to
achieve the transition
state per unit time

16. The unique properties of enzyme catalyst
• Enzyme–substrate complexes:
• Enzymes are specific and efficient catalyst
• Lock and key fit of enzymes and substrates help produce enzyme
specificity.
• Induced fit hypothesis of Koshland gained more acceptance later.

17. Lock-and-key model of enzyme action
In this early model, the substrate (key) was
believed to bind tightly to a site on the enzyme
(lock) that was exactly complementary to it in its
size, shape and charge.
Proposed by Emil Fischer (1894): the shape
of the substrate and the active site of the
enzyme are thought to fit together like a key
into its lock.

18. Enzyme-substrate complexes
Lock and key fit of enzymes and substrates helps produce enzyme specificity: A
substrate molecule must contact an enzyme molecule before it can be transformed into
products. Once the substrate has made contact, it must bind to the enzyme at the active
site— a place where the conversion of substrate to product can take effect. The active
site is usually a dimple, pocket or crevice formed by fold in the chain conformation of the
protein. When the substrate key fits the enzyme lock, an enzyme-substrate complex is
formed.
The fit between enzyme and substrate in binding helps to produce enzyme specificity.
Complementarity between enzyme and and substrate is governed by factors besides
shape– e.g., the electrical charge. The substrate may be positively charged and the
enzyme’s active site negatively charged and vice versa.
Hydrogen bonds and other weak forces may also hold the the enzyme- substrate complex
together. Hydrophobic regions of the substrate associate with similar regions on the
active site of the enzyme. For most enzymes contributions from shape, charge, and a
large number of weak forces are responsible for the formation and strength of the
complex.

19. Induced fit hypothesis
proposed in 1958 by Daniel E. Koshland, Jr.: the binding of substrate induces a
conformational change in the active site of the enzyme. In addition, the enzyme
may distort the substrate, forcing it into a conformation similar to that of the
transition state

20. Transition state stabilization
• Linus Pauling (1946) formulated the principle that
• An enzyme increases the reaction rate not by binding tightly to the
substrates, but by binding tightly to the reaction’s transition state,
i.e., substrate that have been strained toward the structure of the
product. In other word, the tightly bound key of the lock and key
model is the ‘transition state’, not the substrate.
Pauling’s theory of enzyme’s actions is consistent with the chemical
catalytic mechanisms, in which the catalyst stabilizes the transition
state of the reaction. In an enzyme, tight binding( stabilization) of
the transition state occurs in addition to acid-base, covalent, or
metal ion catalysis.

21. Effect of very tight substrate binding on enzyme catalysis
The red line indicates the free
energy changes as the reactants
pass through a transition state
to products. The blue line
indicates free energy changes
for the same reaction when the
reactants (substrates) are very
tightly bound to the enzyme (
they are at a very low free
energy level) before they react.
The activation energy barrier
(G‡) is therefore increased
because the tight binding must
be overcome before the
substrates can reach the
transition state.

22. Temperature Effects
Like most chemical reactions, the rate of an enzyme-catalyzed reaction
increases as the temperature is raised. A ten degree Centigrade rise in
temperature will increase the activity of most enzymes by 50 to 100%.
Variations in reaction temperature as small as 1 or 2 degrees may introduce
changes of 10 to 20% in the results. In the case of enzymatic reactions, this is
complicated by the fact that many enzymes are adversely affected by high
temperatures. As shown in Figure 13, the reaction rate increases with
temperature to a maximum level, then abruptly declines with further increase
of temperature. Because most animal enzymes rapidly become denatured at
temperatures above 40°C, most enzyme determinations are carried out
somewhat below that temperature.

23. Effects of pH
Enzymes are affected by changes in pH. The most favorable pH value
- the point where the enzyme is most active - is known as the
optimum pH. This is graphically illustrated in Figure 14.
Extremely high or low pH values generally result in complete loss of activity for
most enzymes. pH is also a factor in the stability of enzymes. As with activity, for
each enzyme there is also a region of pH optimal stability.
The optimum pH value will vary greatly from one enzyme to another

24. Enzymes use chemical catalytic mechanism
• There are three basic kinds of chemical catalytic
mechanisms used by enzymes:
- Acid-base catalysis
- Covalent catalysis
- Metal ion catalysis

25. Acid-base catalysis
• In acid-base catalysis a proton is transferred between the
enzyme and the substrate
• It may be either acid catalysis or base catalysis or both
• Many enzymes use both

26. Tautomerization of a ketone
Tautomers are interconvertible isomers that differ in the placement of a hydrogen and
a double bond. The dotted lines represent bonds in the process of breaking or
forming.

27. Acid catalysis
If a catalyst (H—A) donates a proton to the to the keton’s oxygen atom, it
reduces the unfavorable character of the transition state, thereby lowers its
energy. Hence, it also lowers the activation energy barriers for the reaction.
This is an example of acid catalyst, since the catalyst acts as an acid by
donating a proton.

28. Keto-enol tautomerization (base catalysis)
The same keto-enol tautomerization reaction shown in the previous slide can be
accelerated by a catalyst that can accept a proton. In this case, it is a base catalyst (:B),
where the dots represent unpaired electron. Base catalysis lowers the energy of
transition state and thereby accelerates the reaction.

29. Amino acid side chains that can act as acid-base catalysts

30. Covalent catalysis…
• In covalent catalysis, a covalent bond is formed between the catalyst and the
substrate during formation of the transition state
• In covalent catalysis, an electron rich group in the enzyme active site forms a
covalent adduct with a substrate. This covalent complex is much more stable
than a transition state and can be isolated.
• Covalent catalysis is also called nucleophilic catalysis, because the catalyst is a
nucleophile (an electron-rich group in search of a electron -poor center ( a
compound with an electron deficiency is known as an electrophile)
• Enzymes that use covalent catalysis undergo a two-part reaction process. So
the reaction coordinate diagram contains two energy barriers with the reaction
intermediate between them.

31. Protein groups that can act as covalent catalysts

32. Reaction coordinate diagram for a reaction accelerated by
covalent catalysis
Two transition
states flank the
covalent
intermediate. The
relative heights of
the activation
energy barriers
(the energies of
the two transition
states, X‡
1 and X‡
2)
vary depending on
the reactions.

33. Metal ion catalysis…
• Metal ions participate in enzymatic reactions by mediating
oxidation-reduction reactions, or by promoting the
reactivity of other groups in the enzyme’s active site
through electrostatic effects
• A protein bound metal ion can interact directly with the
reacting substrate

34. Metal ion catalysis
Example: The conversion of acetaldehyde to ethanol catalyzed by the liver enzyme
alcohol dehydrogenase. Here a zinc ion stabilizes the developing negative charge on the
oxygen atom during formation of the transition state.

35. A note on nomenclature…
• The International Enzyme Commission (EC) has recommended a
systematic nomenclature for enzymes.
• This commission assigns names and numbers to enzymes according to
the reaction they catalyze. An example of systematic enzyme name is
EC 3.5.1.5 urea aminohydrolases for the enzyme that catalyzes the
hydrolysis of urea.
• The name of an enzyme frequently provides a clue to its function. In
some cases, an enzyme is named by incorporating the suffix -ase into
the name of its substrate, e.g., pyruvate decarboxylases catalyzes the
removal of a CO2 group from pyruvate

36. A note on nomenclature
• Certain protein cutting digestive enzymes are exception to this general
rule of enzyme nomenclature, e.g., pepsin, trypsin, chymotrypsin and
thrombin.
Isozyme: Within an organism more than one enzyme may catalyze a
given reaction. Multiple enzymes catalyzing the same reaction is called
isozymes.
- isozymes differ in their catalytic properties. Consequently, the
various isozymes that are present in different tissues or at different
developmental stages can carry out slightly different metabolic
functions.

37. Pyruvate decarboxylase

38. Alanine aminotransferase

39. Enzyme classification
Class of Enzyme Type of reaction catalyzed
1. Oxidoreductases Oxidation-reduction reactions
2. Transferases Transfer of functional groups
3. Hydrolases Hydrolysis reactions
4. Lyases Group elimination to form double bond
5. Isomerases Isomerization reactions
6. Ligases Bond formation couples with ATP hydrolysis