The document provides an overview of enzymology and enzyme catalysis. It discusses: 1. The teaching objectives which are to introduce enzymes as catalysts, classify enzyme types and mechanisms, illustrate enzyme kinetics, highlight mechanisms of enzyme regulation and clinical importance. 2. Key characteristics of enzyme-catalyzed reactions including high specificity and regulation in response to changes. 3. Mechanisms by which enzymes catalyze reactions including proximity and orientation effects, transition state stabilization, and acid/base catalysis.

1. Enzymology

2. My Teaching Objectives
1. Introduce the concept of enzymes as catalysts in terms of their
effects on the activation energy and dynamics of biochemical
reactions
2. Classify enzyme types and mechanisms of enzymatic catalysis
3. Illustrate how enzyme activity can be quantitatively measured
through plots of substrate concentration versus product
formation (enzyme kinetics).
4. Highlight mechanisms relevant in the pharmacological and
physiological regulation of enzyme activity
5. Highlight clinical importance of enzymes

3. Enzymes
 Enzymes are catalysts that have special characteristics to
facilitate the biochemical reactions in the biological systems.
 Enzyme-catalyzed reactions have the following characteristics
in comparison with the general catalyzed reactions:
Common features
 Apply to the thermodynamically allowable reactions
 Reduce the activation energy
 Cannot alter reaction equilibria - only enhance the reaction rates.
 Unchanged after reaction (no changes in quantity and quality before and
after the reactions.
Unique features
 Enzyme- catalyzed reactions have very high catalytic efficiency.
 Enzymes have a high degree of specificity for their substrates.
 Enzymatic activities are highly regulated in response to the external
changes.
 All are proteins- so liable to denaturation

4. High Specificity
Unlike conventional catalysts, enzymes
demonstrate the ability to distinguish
different substrates. There are three types
of substrate specificities.
Absolute specificity
Relative specificity
Stereospecificity
Absolute specificity: Enzymes can
recognize only one type of substrate and
implement their catalytic functions.
O C
NH2
NH2
+ H2O 2NH3 + CO2
urea
urease
O C
NH
NH2
+ H2O
methyl urea
CH3
Relative specificity- Enzymes catalyze
one class of substrates or one kind of
chemical bond in the same type.
protein kinase A
protein kinase C
protein kinase G
To phopharylate the -OH group of serine
and threonine in the substrate proteins,
leading to the activation of proteins.
O
H
OH
H
H
OH
H
OH
CH2OH
H
CH2OH
H
CH2OH
OH H
H OH
O
O
1
1
O
H
OH
H
H
OH
H
OH
CH2
H
CH2OH
H
CH2OH
OH H
H OH
O
O
1
1
O
O
OH
H
H
H
OH
H
OH
CH2OH
H 1
sucrose
raffinose
sucrase
Stereospecificity- enzymes can act on
only one form of isomers of the substrates.
H
C
H3C COOH
OH
H
C
H3C OH
COOH
A
B
C A
B C
Lactate dehydrogenase can recognize only
the L-form but not the D-form lactate.

5. 1. Fragile structures of the living systems
2. Low kinetic energy of the reactants
3. Low concentration of the reactants
4. Toxicity of catalysts
5. Complexity of the biological systems
Chemical reactions in living systems are
quite different from that in the industrial
situations because of
Why Need for special catalysts

6. Enzyme – Terms
• Active site - a region of an enzyme comprised of different amino acids where
catalysis occurs (determined by the tertiary and quaternary structure of each
enzyme)
• Substrate - the molecule being utilized and/or modified by a particular enzyme at its
active site
• Prosthetic group - a metal or other co-enzyme covalently bound to an enzyme.
They supply the active sites with reactive groups not present on the side chains of
AA residues.
• Holoenzyme - a complete, catalytically active enzyme including all co-factors
• Apoenzyme - the protein portion of a holoenzyme minus the co-factors
• Isozyme - (or iso-enzyme) an enzyme that performs the same or similar function of
another enzyme. This generally arises due to similar but different genes encoding
these enzymes and frequently is tissue-type specific or dependent on the growth or
developmental status of an organism.
• Ribozymes - segments of RNA that display enzyme activity in the absence of
protein. Ribozymes have the ability to self-cleave.
Examples: RNase P and peptidyl transferase
• Abzymes - antibodies raised to bind the transition state of a reaction of interest.

7. Enzyme co-factors
Co-factor - chemical species required by inactive apoenzymes (protein only) to convert
themselves to active holoenzymes.
Coenzymes - Act as group-transfer reagents to supply active sites with reactive groups not
present on the side chains of amino acids either cosubstrates or Prosthetic groups.
Metabolite coenzymes: they are synthesized from the common metabolites. several NTP,
ATP (most abundant), UDP-glucose
Vitamin-derived coenzymes: they are derivatives of vitamins, and can only be obtained
from nutrients. NAD and NADP+, FAD and FMN, lipid vitamins, …
Cosubstrates -They substrates in nature. Their structures are altered for subsequent
reactions. Shuttle mobile metabolic groups among different enzyme-catalyzed reactions.
Activator ions: loosely and reversibly bound, often participate in the binding of substrates.
Metal ions of metalloenzymes: tightly bound, and frequently participate directly in catalytic
reactions.
Metal-activated enzymes contain loosely bound metal ions: (usually Na+, K+, Mg+2, or Ca+2)
Metal ions -Transfer electron, Linkage of S and E；Keep conformation of E-S complex and
Neutralize anion.
Activator ions
(loosely bound)
Metal ions of
metalloenzymes
(tightly bound)
Cosubstrates
(loosely bound)
Prosthetic
groups
(tightly bound)
Essential ions Coenzymes
Cofactors

8. Enzyme Classification

9. Catalysts increase product formation by
(1) lowering the energy barrier (activation energy) for the product to form
(2) increases the favorable orientation of colliding reactant molecules for
product formation to be successful (stabilize transition state
intermediate)
Enzyme Reaction Principles

10. Structure of the enzyme active site
• Enzymes are proteins having well defined structures.
• Some functional groups are close enough in space to form a
portion called the active center.
• Active centers look like a cleft or a crevice.
• Active centers are hydrophobic.
• The active center has two essential groups in general.
• Binding group: to associate with the reactants to form an
enzyme-substrate complex
• Catalytic group: to catalyze the reactions and convert
substrates into products

11. Substrates Accommodation at the active site
Lock-and-key model
Both E and S are rigid and fixed, so
they must be complementary to each
other perfectly in order to have a right
match.
The substrate is drawn into a closely
matching cleft on the enzyme
molecule.
Induced-fit model
The binding induces conformational changes of
both E and S, forcing them to get a perfect
match.
Hexokinase, the first enzyme in the glycolysis
pathway, converted glucose to glucose-6-
phosphate with consuming one ATP molecule.
Two structural domains are connected by a
hinge. Upon binding of a glucose molecule,
domains close, shielding the active site for water.

12. Mechanisms of Catalysis at Active Site
• Mechanisms used by enzymes to enhance reaction rates include: (1st 4 mechanisms
based on BINDING of substrate and/or transition state)
1. Proximity & orientation
2. Desolvation (one type of electrostatic catalysis)
3. Preferential binding of the transition state
4. Induced fit
5. General acid/base catalysis
6. Covalent (nucleophilic) catalysis
7. Metal ion catalysis
8. (Electrostatic catalysis)
The chemical mechanism of serine proteases like chymotrypsin illustrates:
– Proximity and orientation
– Transition state stabilization
– Covalent catalysis, involving a “catalytic triad” of Asp, His and Ser in the
active site
– general acid-base catalysis
– electrostatic catalysis

13. Catalysis Mechanisms
• Proximity and orientation
• By oriented binding and
immobilization of the substrate,
enzymes facilitate catalysis by four
ways;
1. bring substrates close to catalytic
residues
2. Binding of substrate in proper
orientation (up to 102-fold)
3. Stabilization of transition state by
electrostatic interactions
4. Freezing out of translational and
rotational mobility of the substrate
(up to 107-fold)
• Transition state binding
• An enzyme may binds the transition
state of the reaction with greater
affinity than its substrate or products.
This together with the previously
discussed factors accounts for the
high rate of catalysis For example, if
enzyme binds the transition state
with 34.2 kJ/mol (= 2 hydrogen
bonds) it results in 106-fold rate
enhancement
• General acid catalysis: Proton transfer from an acid
lowers the free energy of a reaction’s transition state
.Example, keto-enol tautomerization Enhanced by
proton donation or proton abstraction (general base
catalyzed).Asp, Glu, His, Cys, Tyr, Lys have pK’s in or
near the physiological range The ability of enzymes to
arrange several catalytic groups around their substrates
makes concerted acid-base catalysis a common
enzymatic mechanism
• Covalent Catalysis accelerates reaction rates through
the transient formation of a catalyst-substrate covalent
bond. Usually, nucleophilic group on enzyme attacks an
electrophilic group on the substrate = nucleophilic
catalysis Example: decarboxylation of acetoacetate
• Good covalent catalysis must be (i) highly nucleophile
and (ii) form a good leaving group. These are imidazole
and thiol groups, i.e. Lys, His and Cys, Asp, Ser, some
coenzymes (thiamine pyrophosphate, pyridoxal
phosphate)
• Three stages of Covalent Catalysis
• Nucleophilic attack of enzyme on substrate
• Withdrawal of electrons
• Elimination of catalysts by reversion of step 1

14. Transition state stabilization
1. No catalysis is obtained by just binding substrate tightly!-A protein that binds substrate very tightly, by
being complementary to the substrate, will NOT lower the free energy of activation for the reaction. It can, in
fact, actually make the reaction less likely to occur since the energy difference between ES and the transition
state may become greater than that between S and the transition state. As a result, such a protein will not act
as an enzyme (a/b).
2. A protein that is complementary to the transition state, such that it binds the transition state much more tightly
than it binds substrate, will reduce the free energy of the transition state but will not substantially change the
free energy of ES. As a result, the activation energy is dramatically reduced so that the rate constant is
increased and reaction is catalyzed (c).

15. Effect of Temperature
Enzymes often have a
narrow range of
conditions under which
they operate properly.
For most plant and animal
enzymes, there is little
activity at low
temperatures.
Enzyme activity increases
with temperature, until the
temperature is too high for
the enzyme to function.
(See diagram right).
At this point, enzyme
denaturation occurs and the
enzyme can no
longer function.
Rate
of
reaction
Temperature
(°C)
Too cold for the
enzyme to
operate
Optimum temperature
for the enzyme
Rapid
denaturation
at high
temperatures

16. Effect of pH
Enzymes can be affected by pH.
Extremes of pH (very acid or
alkaline) away from the enzyme
optimum can result in
enzyme denaturation.
Enzymes are found in very
diverse pH conditions, so they
must be suited to perform in these
specialist environments.
Pepsin is a stomach enzyme and has
an optimal working pH of 1.5,
which is suited for the very acidic
conditions of the stomach.
Urease breaks down urea and has an
optimal pH of near neutral. See
diagram right.
Enzymes often work over a range of pH
values, but all enzymes have an optimum
pH where their activity rate is fastest.
Pepsin Urease Trypsin
Enzyme
activity
pH
Alkaline
Acid
1 3
2 4 5 6 7 8 9 10

17. Factors Affecting Enzyme Reaction
Rates
Enzyme concentration
Rate
of
reaction
Effect of Enzyme
Concentration
Rate of reaction continues to increase
with an increase in enzyme concentration.
This relationship assumes non-limiting
amounts of substrate and cofactors.
Concentration of substrate
Effect of Substrate
Concentration
Rate of reaction increases and then plateaus
with increasing substrate concentration.
This relationship assumes a fixed amount
of enzyme.

18. Enzyme Kinetics Equation

19. Michaelis-Menten Equation

20. Initial Velocity (vo) and [S]
• The concentration of substrate [S] present will greatly influence the rate of product
formation, termed the velocity (v) of a reaction. Studying the effects of [S] on the
velocity of a reaction is complicated by the reversibility of enzyme reactions, e.g.
conversion of product back to substrate. To overcome this problem, the use of initial
velocity (vo) measurements are used. At the start of a reaction, [S] is in large
excess of [P], thus the initial velocity of the reaction will be dependent on substrate
concentration
• When initial velocity is plotted against [S], a hyperbolic curve results, where Vmax
represents the maximum reaction velocity. At this point in the reaction, if [S] >> E, all
available enzyme is "saturated" with bound substrate, meaning only the ES complex
is present.
Michaelis-Menten Curve

21. Steady State Assumption
• The M-M equation was derived in part by making several
assumptions. An important one was: the concentration of
substrate must be much greater than the enzyme
concentration. In the situation where [S] >> [E] and at
initial velocity rates, it is assumed that the changes in the
concentration of the intermediate ES complex are very
small over time (vo). This condition is termed a steady-
state rate, and is referred to as steady-state kinetics.
Therefore, it follows that the rate of ES formation will
be equal to the rate ES breakdown.

22. Meaning of Km
• An important relationship that can be derived from the Michaelis-Menten
equation is the following: If vo is set equal to 1/2 Vmax, then the relation
Vmax /2 = Vmax[S]/Km + [S] can be simplied to Km + [S] = 2[S], or Km = [S].
This means that at one half of the maximal velocity, the
substrate concentration at this velocity will be equal to the
Km. This relationship has been shown experimentally to be valid for many
enzymes much more complex in regards to the number of substrates and
catalytic steps than the simple single substrate model used to derive it.
• The significance of Km will change based on the different rate constants and
which step is the slowest (also called the rate-limiting step). In the simplest
assumption, the rate of ES breakdown to product (k2) is the rate-determining
step of the reaction, so k-1 >> k2 and Km = k-1/k1. This relation is also
called a dissociation constant for the ES complex and can be used as a
relative measure of the affinity of a substrate for an enzyme (identical to Kd).
However if k2 >> k-1 or k2 and k-1 are similar, then Km remains more
complex and cannot be

23. Uses of Km
• Experimentally, Km is a useful parameter for characterizing the
number and/or types of substrates that a particular enzyme will
utilize.
• It is also useful for comparing similar enzymes from different
tissues or different organisms.
• Also, it is the Km of the rate-limiting enzyme in many of the
biochemical metabolic pathways that determines the amount of
product and overall regulation of a given pathway.
• Clinically, Km comparisons are useful for evaluating the effects
mutations have on protein function for some inherited genetic
diseases.
• Km is independent of [E]. It is determined by the structure of E,
the substrate and environmental conditions (pH, T, ionic
strength, …)
•

24. Meaning of Vmax
• The reaction velocity of an enzymatic reaction when the binding
sites of E are saturated with substrates.
• It is proportional to [E].
• The values of Vmax will vary widely for different enzymes and
can be used as an indicator of an enzymes catalytic efficiency.
It does not find much clinical use.
• There are some enzymes that have been shown to have the
following reaction sequence:
• In this situation, the formation of product is dependent on the
breakdown of an enzyme-product complex, and is thus the rate-
limiting step defined by k3.

25. Derivation of kcat
• A more general term has been defined, termed
kcat, to describe enzymes in which there are
multiple catalytic steps and possible multiple
rate-limiting steps. The Michaelis-Menten
equation can be substituted with kcat

26. Definition and Use of kcat
• The constant, kcat (units of sec-1), is also called
the turnover number because under saturating
substrate conditions, it represents the number
of substrate molecules converted to product in
a given unit of time on a single enzyme
molecule. In practice, kcat values (not Vmax) are
most often used for comparing the catalytic
efficiencies of related enzyme classes or
among different mutant forms of an enzyme.

27. Limitations of M-M
1. Some enzyme catalyzed rxns show more complex behavior
E + S<->ES<->EZ<->EP<-> E + P
With M-M can look only at rate limiting step
2. Often more than one substrate
E+S1<->ES1+S2<->ES1S2<->EP1P2<-> EP2+P1<-> E+P2
Must optimize one substrate then calculate kinetic
parameters for the other
3. Assumes k-2 = 0
4. Assume steady state conditions

28. Two Substrate Reactions
• Many enzyme reactions involve two or more
substrates. Though the Michaelis-Menten equation
was derived from a single substrate to product
reaction, it still can be used successfully for more
complex reactions (by using kcat).
Random
Ordered
Ping-pong

29. Two Substrate Reactions (cont)
• In random order reactions, the two substrates do not bind to
the enzyme in any given order; it does not matter which binds
first or second.
• In ordered reactions, the substrates bind in a defined
sequence, S1 first and S2 second.
• These two reactions share a common feature termed a ternary
complex, formed between E, ES1, ES2 and ES1S2. In this
situation, no product is formed before both substrates bind to
form ES1S2.
• Another possibility is that no ternary complex is formed and the
first substrate S1 is converted to product P1 before S2 binds.
These types of reactions are termed ping-pong or double
displacement reactions.

30. Lineweaver-Burke Plots
(double reciprocal plots)
•Plot 1/[S] vs 1/Vo
•L-B equation for straight
line
•X-intercept = -1/Km
•Y-intercept = 1/Vmax
•Easier to extrapolate
values w/ straight line vs
hyperbolic curve

31. B
B
B
B
B B
B
0
0.05
0.1
0.15
0.2
0.25
0 1 2 3 4 5 6 7 8 9 10
Vo
[S]
[S] Vo
0.5 0.075
0.75 0.09
2 0.152
4 0.196
6 0.21
8 0.214
10 0.23
V max
Km
Km ~ 1.3 mM
Vmax ~ 0.25

32. Uses of double reciprocal plot
• The x intercept value is equal to -1/Km.
The biggest advantage to using the
double reciprocal plot is a more
accurate determination of Vmax, and
hence Km. It is also useful in
characterizing the effects of enzyme
inhibitors and distinguishing between
different enzyme mechanisms.

33. Enzyme Inhibitor Types
• Inhibitors of enzymes are generally molecules
which resemble or mimic a particular enzymes
substrate(s). Therefore, it is not surprising that
many therapeutic drugs are some type of
enzyme inhibitor. The modes and types of
inhibitors have been classified by their kinetic
activities and sites of actions. These include
Reversible Competitive Inhibitors, Reversible Non-
Competitive Inhibitors, and Irreversible Inhibitors

34. Enzyme Inhibition
• Inhibitor – substance that binds to an enzyme and interferes
with its activity
• Can prevent formation of ES complex or prevent ES breakdown
to E + P.
• Irreversible and Reversible Inhibitors
• Irreversible inhibitor binds to enzyme through covalent bonds
(binds irreversibly)
• Reversible Inhibitors bind through non-covalent interactions
(disassociates from enzyme)
• Why important?

35. Types of Reversible Enzyme Inhibitors
E + S <-> ES -> E + P
E + I <-> EI
Ki = [E][I]/[EI]
• Competitive
• Uncompetitive
• Non-competitive

36. Competitive Inhibitor (CI)
• CI binds free enzyme
• Competes with substrate for enzyme binding.
• Raises Km (due to the competition for the binding sites) without
effecting Vmax. Km rises, equivalent to the reduction of the affinity.
• Can relieve inhibition with more S- Inhibition depends on the affinity of
enzymes and the ratio of [E] to [S].
V =
Vmax [S]
Km(1 + + [S]
Ki
[ I ]
)
Vmax
1
=
Km 1
[S]
+
Vmax
1
(1 + )
V
[ I ]
Ki

37. Competitive Inhibitors look like substrate
NH2
C
O
HO
NH2
S
O
H2N
O
PABA Sulfanilamide
FH4 (tetrahydrofolate) is a coenzyme in the nucleic acid synthesis, and FH2 (dihydrofolate) is the
precursor of FH4. Bacteria cannot absorb folic acid directly from environment.
Bacteria use p-amino-benzoic acid (PABA), Glu and dihydropterin to synthesize FH2.
Sulfanilamide derivatives share the structural similarity with PABA, blocking the FH2 formation as a
competitive inhibitor. Glu
H2N COOH
dihydropterin
FH2
synthetase
FH2 FH4
H2N SO2NHR
Sulfanilamide
Methotrexate
PABA FH2
reductase
+
+
COOH
H2C
COOH
malonic acid
HC
COOH
CH
HOOC
succinate
succinate
dehydrogenase
fumaric acid
CO-COOH
H2C
COOH
oxaloacetate
H2C
COOH
CH2
HOOC

38. Uncompetitive Inhibitor (UI)
•UI binds ES complex
•Prevents ES from proceeding to E + P or back to E + S.
•Lowers Km & Vmax, but ratio of Km/Vmax remains the same
•Occurs with multisubstrate enzymes
Vmax
1
=
Km 1
[S]
+
Vmax
1
V
(1 + )
[ I ]
Ki

39. Non-competitive Inhibitor (NI)
• NI can bind free E or ES complex. Inhibitors bind to other sites rather than
the active sites on the free enzymes or the E-S complexes. The E-I complex
formation does not affect the binding of substrates. The E-I-S complexes do
not proceed to form products.
• Lowers Vmax, but Km remains the same since NI’s don’t bind to S binding
site therefore don’t effect Km
• Alters conformation of enzyme to effect catalysis but not substrate binding
Vmax
1
=
Km 1
[S]
+
Vmax
1
(1 + )
V
[ I ]
Ki
(1 + )
[ I ]
Ki

40. Summary of inhibition

41. • Irreversible inhibitors very tightly bound to enzyme, either covalently or noncovalently, but effectively don't
come off.
• Main categories include:
– Group-specific covalent modifying agents- they react with specific type of enzyme functional group
(e.g., Ser-OH, or Cys-SH, or His imidazole) on any enzyme/protein example is
Diisopropylphosphofluoridate (DIPF), potent nerve gas (poison). This reacts with specific, reactive Ser-
OH on many enzymes example: reaction with reactive, catalytic OH group of acetyl- cholinesterase at
synaptic junctions. The modified enzyme is inactive
– Affinity labels -structural similarity to substrate "guides" reagent to active site. The reaction at active site
covalently inactivates enzyme. Example: Tosyl phenylalanyl chloromethylketone (TPCK) whose phenyl
group binds in substrate specificity site of chymotrypsin
– Transition state analogs -structurally similar to transition state, which binds even more tightly to
enzyme than substrate binds, so very high affinity for active site.
• Transition state analogs useful for:
• understanding catalytic mechanisms (clues about structure of transition state)
• very specific inhibitors of enzymes (pharmaceutical applications)
• antigens for immunizing lab animals to generate antibodies with binding sites complementary to the
transition state such that the antibodies themselves have catalytic activity ("abzymes")
– Suicide inhibitors (mechanism-based inhibitors) - Structural similarity to substrate "guides" reagent
to active site. The enzyme treats it as a substrate, starting chemical catalytic process with inhibitor and
the chemical mechanism itself leads enzyme to react covalently with inhibitor, thus "committing suicide”.
Mechanism-based inhibition depends on chemical mechanism of catalysis. Example: penicillin (inhibits
an enzyme, a transpeptidase, required for bacterial cell wall synthesis)
Irreversible Inhibitors

42. Irreversible Inhibitor: Allopurinol

43. Diisopropyl Phosphofluoridate: Irreversible
Acetylcholinesterase Inhibitor (Example)

44. Irreversible Inhibitor: Penicillin (Ex)

45. Pharmaceutically important enzyme
inhibitors -Penicillin (an antibiotic)
• both a transition-state analog and a suicide substrate
• covalently inhibits a transpeptidase (enzyme) involved in bacterial cell wall
synthesis (eukaryotic cells don't have this enzyme)
• Normal transpeptidase catalytic mechanism: nucleophilic attack of enzyme
Ser–OH on substrate, making a covalent acyl-enzyme intermediate
• Covalent intermediate continues in enzyme-catalyzed reaction to form
peptide cross-link in peptidoglycan structure of cell wall, regenerating free
enzyme for another round of catalysis.
• Penicillin resembles transition state in structure, so penicillin a) binds very
tightly and b) is very reactive.
• Normal catalytic mechanism makes covalent intermediate with penicillin, but
enzyme-penicillin derivative can't continue.
• Inhibitor is "stuck" on enzyme (covalently attached), and modified enzyme is
now inactive because of its own catalytic activity -- it committed suicide!

46. Pharmaceutically important enzyme
inhibitors -NSAIDS
• Aspirin (acetylsalicylate), a non-steroidal anti-
inflammatory drug (NSAID)
• covalently (irreversibly) inactivates enzyme
(PGH2 synthase, cyclooxygenase activity, also
known in its two forms as COX 1 and COX 2)
involved in prostaglandin biosynthesis
• anti-inflammatory action due to blocking of
prostaglandin synthesis
• covalently modifies (acetylates) specific Ser-
OH group in channel through which substrate
(arachidonic acid, a 20-C fatty acid) must pass
to reach active site; NSAIDs block active site
access, inhibiting enzyme, preventing
prostaglandin synthesis, reducing
inflammation.
• Aspirin also reduces blood clotting because
same enzyme is also needed for synthesis of
thromboxane A2 (TXA2), involved in blood
platelet aggregation in clotting.
• Other NSAIDs (nonsteroidal anti-
inflammatory agents) besides aspirin:
e.g., ibuprofen (=Motrin, Advil),
acetaminophen (=Tylenol),
indomethacin, naproxen (=Aleve)
• competitive (reversible) inhibitors of
cyclooxygenase activity of PGH2
synthase
• block prostaglandin synthesis and thus
act as anti-inflammatory agents
• reversibly bind in channel through which
substrate must access enzyme active
site, so act as competitive inhibitors by
preventing substrate binding, even
though they don’t bind IN the active site.
• (Aspirin inhibits same enzyme
irreversibly, by acetylating Ser-OH group
in "entrance" channel to active site, but
not actually in active site.)

47. More Examples of Pharmaceutically
important enzyme inhibitors
• Statins –Inhibitors of HMG-CoA reductase, the rate-limiting, control enzyme in
cholesterol biosynthesis. Competitive inhibitors of HMG-CoA reductase are
cholesterol-lowering drugs (decrease rate of cellular cholesterol
biosynthesis).Structures similar to substrate for HMG-CoA reductase (mevalonate) –
e.g., Mevacor (lovastatin), Pravachol (pravastatin), and Zocor (simvastatin)
• Ethanol- Used as an antidote for ethylene glycol (antifreeze) or methanol (wood
alcohol) poisoning. The toxic effects of ethylene glycol and of methanol depend on
their -OH groups being oxidized to aldehyde (by alcohol dehydrogenase in body) and
then to carboxylic acids. Ethanol (another substrate with less toxic oxidation products)
competes for binding to alcohol dehydrogenase. If alcohol dehydrogenase molecules
are all occupied with ethanol as a substrate, ethylene glycol (or methanol) passes
through body without being oxidized and is excreted (kidneys)
• anti-HIV drugs (anti-AIDS)
– AZT: metabolized to AZT-triphosphate, which terminates growing DNA chains
in reaction catalyzed by HIV viral reverse transcriptase; much higher affinity for
HIV reverse transcriptase than for cellular DNA polymerases.
– Saquinavir and Ritonavir: VERY tight-binding inhibitors (transition state
analogs) of HIV protease (enzyme needed to process large HIV polyprotein
precursors to release viral proteins)

48. Definition of Ki
• For reversible inhibitors, a term Ki can be
determined.
• For competitive inhibitors, the following relation
can be used: Km + I = Km (1 + [I] / Ki ) ;
(where Km + I is the determined Km in the
presence of [I]).
• Determining the Ki for other inhibitor types is
related but much more complex and not within
the scope of this lecture or course

49. Uses of Ki
• Ki values are used to characterize and compare the
effectiveness of inhibitors relative to Km. This
parameter is especially useful and important in
evaluating the potential therapeutic value of inhibitors
(drugs) of a given enzyme reaction. For example, Ki
values are used for comparison of the different types
of HIV protease inhibitors. In general, the lower the
Ki value, the tighter the binding, and hence the
more effective an inhibitor is.

50. • Enzymatic activity is a measure of the capability of an enzyme
of catalyzing a chemical reaction.
• It directly affects the reaction rate.
• International unit (IU): the amount of enzyme required to
convert 1 µmol of substrate to product per minute under a
designated condition.
• Determination of the enzymatic activity requires proper
treatment of enzymes, excess amount of substrate, optimal T
and pH, …
• One katal is the amount of enzyme that converts 1 mol of
substrate per second.
• IU = 16.67×10-9 kat
Enzymatic activity

51. • Many biological processes take place at a
specific time; at a specific location and at a
specific speed.
• The catalytic capacity is the product of the
enzyme concentration and their intrinsic
catalytic efficiency.
• The key step of this process is to regulate
either the enzymatic activity or the enzyme
quantity.
Enzyme regulation

52. • Maintenance of an ordered state in a timely
fashion and without wasting resources
• Conservation of energy to consume just
enough nutrients
• Rapid adjustment in response to environmental
changes
Reasons for regulation

53. • Constitutive enzymes (house-keeping):
enzymes whose concentration essentially remains
constant over time
• Adaptive enzymes: enzymes whose quantity
fluctuate as body needs and well-regulated.
• Regulation of enzyme quantity is accomplished
through the control of the genes expression.
Regulation of Enzyme Quantity

54. • Inducer: substrates or structurally related compounds that can initiate
the enzyme synthesis
• Stimulation of Enzyme Synthesis in response to an inducer .e.g.
glucokinase is stimulated by glucose while insulin induces the
synthesis of glycokinase enzyme after a carbohydrate meal.
• Repressor: compounds that can curtail the synthesis of enzymes in an
anabolic pathway in response to the excess of an metabolite
• Repression- Inhibition of Enzyme Synthesis in response to an
repressor e.g.
– the hormone glucagon represses glukokinase during starvation
– Product of enzyme- heme represses ALA synthase (heme synthesis)
• Both are cis elements, trans-acting regulatory proteins, and specific
DNA sequences located upstream of genes
Controlling the synthesis

55. • Enzymes are immortal, and have a wide range of lifetime. LDH4 5-6
days, amylase 3-5 hours.
• They degrade once not needed through proteolytic degradation.
• The degradation speed can be influenced by the presence of ligands
such as substrates, coenzymes, and metal ions, nutrients and
hormones.
Controlling the degradation

56. • Lysosomic pathway:
– Under the acidic condition in lysosomes
– No ATP required
– Indiscriminative digestion
– Digesting the invading or long lifetime proteins
• Non-lysosomic pathway:
– Digest the proteins of short lifetime
– Labeling by ubiquitin followed by hydrolysis
– ATP needed
Degradation pathway

57. Enzymes/pathways in cellular
organelles
organelle Enzyme/metabolic pathway
Cytoplasm Aminotransferases, peptidases, glycolysis, hexose monophosphate shunt,
fatty acids synthesis, purine and pyrimidine catabolism
Mitochondria Fatty acid oxidation, amino acid oxidation, Krebs cycle, urea synthesis,
electron transport chain and oxidative phosphorylation
Nucleus Biosynthesis of DNA and RNA
Endoplasmic
reticulum
Protein biosynthesis, triacylglycerol and phospholipids synthesis, steroid
synthesis and reduction, cytochrome P450, esterase
Lysosomes Lysozyme, phosphatases, phospholipases, proteases, lipases, nucleases
Golgi apparatus Glucose 6-phosphatase, 5’-nucleotidase, glucosyl- and galactosyl-
transferase
Peroxisomes Calatase, urate oxidase, D-amino acid oxidase, long chain fatty acid
oxidase

58. 5 principal ways protein/enzyme activity is regulated
1. Allosteric control -Regulation of binding affinity for ligands, and/or of catalytic activity, by
conformational changes caused by binding of the same or other ligands at other sites on
protein ("allosteric effects"). The changes involve simple association/dissociation of small
molecules, so enzyme can cycle rapidly between active and inactive (or more and less
active) states.
2. Multiple forms of enzymes (isozymes) = multiple forms of enzyme that catalyze same
reaction but are products of different genes (so different amino acid sequences) •
Isozymes differ slightly in structure, and kinetic and regulatory properties are different. •
Can be expressed in different tissues or organelles, at different stages of development,
etc.
3. Protein-Protein interaction - Binding of a different protein to the enzyme alters the
enzyme activity (activates or inhibits the enzyme), usually by causing conformational
change.
4. Reversible covalent modification- Modification of catalytic or other properties of
proteins by enzyme catalyzed covalent attachment of a modifying group. The
modifications is removed by catalytic activity of a different enzyme, so enzyme can cycle
between active and inactive (or more and less active) states.
5. Proteolytic activation (Irreversible covalent modification)- Irreversible cleavage of
peptide bonds to convert inactive protein/enzyme to active form. The Inactive precursor
protein = a zymogen (a proenzyme).Proteolytic activation irreversible, but eventually the
activated protein is itself proteolyzed, or sometimes a tight-binding specific inhibitory
protein inactivates it.

59. Allosteric Regulation
• Multisubunit enzymes (more than one active site per enzyme)
• Regulation of binding affinity for ligands (like substrates) and/or catalytic activity (kcat)
• Conformational changes linked with ligand binding
• homotropic effects: binding of "primary" ligand (substrate for an enzyme, O2 for
hemoglobin, etc.) can alter affinity of other binding sites on molecule for that same ligand
• heterotropic effects: binding of other ligands (regulatory signaling molecules), to different
sites from the primary ligand ("regulatory sites") can cause conformational changes that
alter primary ligand binding affinity or catalytic activity
• Sometimes regulatory sites are on different subunits (“regulatory subunits”) from binding
sites for primary ligand.
• Ligand binding-induced conformational changes:
• Ligand concentration = signal (cell needs more or less of some metabolic product)
• Signal detected by regulated enzyme
• Allosteric regulation permits rapid cycling of enzyme between more active and less active
conformations (just association/dissociation of small molecules).
• Allosteric activators ---> higher activity
• Homotropic effector/modulator (substrate itself)
• Heterotropic effectors, e.g. – Metabolite earlier than substrate in same pathway
(“feed-ahead activation”) – Other metabolites (ligands) that act as indicator(s) of
metabolic need
• Allosteric inhibitors ---> lower activity
• Heterotropic effectors/modulators – Product of whole pathway (“feedback
inhibition”) – another ligand that acts as indicator that cell needs less of that
pathway’s product

60. Models for Allosteric Modulation- Symmetry
Model
• Monod, Wyman, Changeux (MWC)
Model: allosteric proteins can exist
in two states: R (relaxed) and T (taut)
• In this model, all the subunits of an
oligomer must be in the same state
• T state predominates in the absence
of substrate S
• S binds much tighter to R than to T
• Cooperativity is achieved because S
binding increases the population of
R, which increases the sites available
to S
• Ligands such as S are positive
homotropic effectors
• Molecules that influence the binding
of something other than themselves
are heterotropic effectors

61. Allosteric T to R transition
Concerted
model
Sequential
model
ET-I ET ER ER-S
I
I S
S

62. Glycogen Phosphorylase
Both Allosteric Regulation and covalent modification
• GP cleaves glucose units from non-reducing ends of glycogen
• A phosphorolysis reaction
• Muscle GP is a dimer of identical subunits, each with PLP covalently linked
• There is an allosteric effector site at the subunit interface
• Pi is a positive homotropic effector
• ATP is a feedback inhibitor, and a negative heterotropic effector
• Glucose-6-P is a negative heterotropic effector (i.e., an inhibitor)
• AMP is a positive heterotrophic effector (i.e., an activator)

63. Glycogen Synthase Regulation: Both
Allosteric and Covalent
PP1: Protein
Phosphatase-1

64. Regulation of Enzyme Activity
Biochemical regulation- Feedback Allosteric Regulation
• 1st committed step of a biosynthetic pathway or enzymes at pathway branch
points often regulated by feedback inhibition.
• Efficient use of biosynthetic precursors and energy
• Accumulation of end products leads to heterotropic allosteric inhibition of the
first enzyme.
B A C
1 3”
3’
2
E F G
4’ 5’
H I J
4” 5”
X
X
Phosphofructokinase( PFK)
Fructose-6-P + ATP -----> Fructose-1,6-bisphosphate + ADP
•PFK catalyzes 1st committed step in glycolysis (10 steps total)
(Glucose + 2ADP + 2 NAD+ + 2Pi → 2pyruvate + 2ATP + 2NADH)
•Phosphoenolpyruvate is an allosteric inhibitor of PFK
•ADP is an allosteric activator of PFK

65. Properties of Allosteric enzymes
1. Catalyze essentially irreversible reactions; are rate limiting
2. Generally contain more than one polypeptide chain
3. Do not follow Michaelis-Menten Kinetics
4. Are regulated by allosteric activators or inhibitors
5. Can be up-regulated by allosteric activators at constant [S]
6. Can be down regulated by allosteric inhibitors at constant [S]
7. Activators and Inhibitors need not have any structural resemblance to substrate
structure Effect of allosteric activators and inhibitors on rate at cellular
concentration of the substrate
Sigmoid kinetics for allosteric enzymes

66. Kinetics of Allosteric Enzymes - Terms
• Cooperativity - in relation to multiple subunit enzymes, changes in the
conformation of one subunit leads to conformational changes in adjacent
subunits. These changes occur at the tertiary and quaternary levels of
protein organization and can be caused by an allosteric regulator.
• Homotropic regulation - when binding of one molecule to a multi-subunit
enzyme causes a conformational shift that affects the binding of the same
molecule to another subunit of the enzyme.
• Heterotropic regulation - when binding of one molecule to a multi-subunit
enzyme affects the binding of a different molecule to this enzyme (Note:
These terms are similar to those used for oxygen binding to hemoglobin)

67. Allosteric Enzymes - Kinetics
• Allosteric enzymes do exhibit saturation kinetics at high [S], but they have a
characteristic sigmoidal saturation curve rather than hyperbolic curve when
vo is plotted versus [S] (analogous to the oxygen saturation curves of
myoglobin vs. hemoglobin). Addition of an allosteric activator (+) tends to
shift the curve to a more hyperbolic profile (more like Michaelis-Menten
curves), while an allosteric inhibitor (-) will result in more pronounced
sigmoidal curves. The sigmoidicity is thought to result from the
cooperativity of structural changes between enzyme subunits (again similar
to oxygen binding to hemoglobin). NOTE: A true Km cannot be determined
for allosteric enzymes, so a comparative constant like S0.5 or K0.5 is used.
Vo vs [S] for Allosteric Enzymes

68. Regulation by Reversible Covalent modification
•Modification of catalytic or other properties of
proteins by covalent attachment of a modifying
group
• Modification reaction catalyzed by a specific
enzyme.
•modifying group removed by catalytic activity of a
different enzyme
•Addition of a group to the enzyme protein by
covalent bond Or removal of a group by cleaving
the covalent bond.
•Reversible-Enzyme can cycle between active and
inactive (or more and less active) states.
•allosteric regulation: "instant" sensing of local
concentration signals, so rapid activity changes
•covalent modifications: generally cause slower and
longer-lasting effects than from allosteric
regulation, with coordinated systemic effects
(e.g., a hormone can trigger covalent modification
events that change activities of metabolic
enzymes in a variety of tissues and many cells.)
• Activities of modifying/ demodifying enzymes
themselves are regulated, allosterically (making
process sensitive to changes in concentration of
small molecules that act as "signals"), or by another
reversible covalent modification process, or both.
•Covalent modification freezes enzyme T or R
conformation
Common forms of revesible covalent modification are;
• Phosphorylation/ dephosphorylation
• Adenylation
• Uridylylation
• ADP-Ribosylation
• Methylation
• Myristoylation
• Acetylation

69. Phosphorylation /Dephosphorylation
•Amino acids with –OH groups are targets for phosphorylation
•Phosphates are bulky (-) charged groups which effect conformation
• probably the most common means of regulating enzymes, membrane
channels, virtually every metabolic process in eukaryotic cells
• Phosphorylation
•Kinase: Any enzyme catalyzing phosphoryl transfer involving ATP or
other nucleoside triphosphate
– named for molecule that "receives" phosphate group e.g.,
– hexokinase transfers terminal phosphate from ATP to a variety of
hexose sugars like glucose (→ glucose-6-phosphate).
• General reaction catalyzed by kinases:
• (target) R-OH + ATP <==> R-OPO3
2– + ADP
•Product = phosphate ester of the target OH group.
• protein kinase: a generic term for kinases that transfer phosphoryl group
from ATP to a PROTEIN (to a Ser-OH, Thr-OH, or Tyr-OH group on the
target protein)
•Phosphorylation of enzymes is catalyzed by protein kinases.
•Dephosphorylation – phosphate group removed by hydrolysis of
phosphate ester (transfer of phosphate to H2O)
•Dephosphorylation of enzymes is catalyzed by a specific PROTEIN
phosphatase.

70. Protein- Protein Interaction (Regulation by
Interaction with regulatory proteins)
• Regulatory proteins are allosteric effectors that can either activate or
inhibit enzyme to which they bind
• Example 1: Protein Kinase A (PKA) (inactivated by binding R
subunits)
• Example 2: Ca2+-Calmodulin – Ca2+ a ubiquitous cytosolic
messenger (signaling molecule).
• Ca2+ concentration "sensed" by Ca2+-binding proteins that
communicate signal to other proteins by protein-protein interactions.
Examples:
• Calmodulin (CaM)
• Troponin C (TnC, protein homologous to CaM in muscle cells,
regulating contraction in response to Ca2+)
– Calmodulin (CaM; Mr 17,000):
– example of a [Ca2+]-sensing protein
– changes conformation when it binds Ca2+
– In Ca2+-bound form, CaM binds to and regulates activities of many
CaM dependent proteins -- enzymes, pumps, etc.

71. cAMP Controls Protein Kinase A Activity
R C
R C
R
R
A
A
A
A
A
A
A
A
C
C
Regulatory
subunits
Catalytic
subunits
cAMP
Active kinase
C
CREB
CREB
P
Nucleus
Activation
Gene
expression
DNA

72. Multienzyme complex- FAS
• Coordinate control
of enzyme complex
• Conformational
change in one
compartment is
transmitted by
protein-protein
interaction to other
components of the
complex amplifying
regulatory effect
• Fatty acid synthase
enzyme complex is
a dimer of two
isentical
polypeptides, each
containing 7
enzymes required
for fatty acid
synthesis
• This inhibits
accumulation of
free intermediates
while allowing for
coordinated control
of enzyme complex

73. Enzyme Regulation by Proteolytic cleavage (irreversible
covalent modification) of proenzyme(zymogen)
• Some enzymes biosynthesized as catalytically inactive precursor polypeptide chains.
• Precursors fold in 3 dimensions
• Later activated by enzyme-catalyzed cleavage (hydrolysis) of 1 or more specific peptide bonds
• ZYMOGENS (or proenzymes): inactive precursors
• zymogen activation: cleavage/activation process Examples:
Proinsulin to Insulin

74. Zymogen Activation in Blood Clotting
•Clotting involves series of
zymogen activations
•Seven clotting factors are serine
proteases involved in clotting
cascade reactions
•Progressive activation of whole
series of clotting factors (named
with Roman numerals in order of
discovery, not in order in which
they work in cascade)
•2 cascades that converge into
final common pathway
•product of “final common
pathway” = fibrin clot
•Very rapid process, huge
amplification of original signal →
enormous response
•Genetic deficiency in any 1
clotting factor = haemophilia
X
X
X X
X
X

75. Blood Clotting
Steps in blood clotting understood at molecular level (no details here)
1. Thrombin-catalyzed proteolysis of fibrinogen → soluble fibrin monomers
2. Self-association of fibrin monomers → insoluble protofibrils (“soft clots”).
3. Covalent cross-linking of fibrin protofibrils → final clot
Why does vitamin K deficiency lead to slow blood clotting?
• Vitamin K required for activity of glutamate carboxylase (make Gla)
• Post-translational modification of specific Glu residues in 4 of the clotting factors → γ-
carboxyglutamate (addition of carboxyl group)
• Multiple Gla residues in zymogens, close in primary structure, bind Ca2+ ions.
• Ca2+ complexes bind neg. charged phospholipids on platelet surfaces.
• Zymogen(Gla)-Ca2+-platelet complexes hold and orient clotting factors in exact
location where they need to be activated (where platelets have bound to injured blood
vessel wall and clot is needed).
• Thus high concentration of active thrombin forms at wound site.
• Vitamin K analogs:
• Coumarins (e.g., dicoumarol) = drugs (anti-clotting agents, anticoagulants) to prevent
heart attacks and strokes
• Don’t inhibit carboxylase -- inhibit another enzyme needed to recycle vitamin K for
repeated use
• Warfarin: analog used as a rat poison

76. Regulation of Blood Clotting and Removal of
clot itself
Regulation of blood Clotting
• Too little or too slow clotting --> haemorrhage (potentially fatal)
• Too much or inappropriately located clots --> heart attacks, strokes (thrombosis, also
potentially fatal)
• Requirements:
• Clots have to form rapidly.
• Clots have to be localized at site of injury.
• Clotting factors have to be removed quickly after clot formation.
• Termination of clotting cascade Removal of clotting factors:
• Dilution by blood flow
• Removal by liver
• Protease degradation (e.g., protease C, activated by thrombin, so final steps in
clot formation also prevent spread of clotting beyond wound area)
• Binding to specific inhibitors (e.g. antithrombin III, a protease inhibitor in blood
plasma, binds thrombin and other serine proteases in clotting cascade tightly in
presence of heparin, a negatively charged polysaccharide; heparin binding →
conformational change in antithrombin III that increases rate of binding to clotting
factors it inhibits.

77. Regulation of Blood Clotting and Removal of
clot itself
Removal of clot itself
• Tissue-type plasminogen activator (TPA), a serine protease – Has a fibrin binding
domain that targets it to fibrin clots, where it finds plasminogen
• Plasminogen binds to fibrin clots, too.
• TPA cleaves plasminogen --> active plasmin (another serine protease)
• Clots dissolved by plasmin, which cleaves fibrin in clots Gene for TPA has been
cloned, used for producing TPA in cultured mammalian cells
• IV administration of TPA within an hour of clot formation in a coronary artery
(heart attack) markedly increases patient’s chance of survival.

78. Enzyme Compartmentation
Molecular Scaffolds – Thought to play three prominent roles:
1. Maintain the specificity of a catalytic pathway. Also referred to as “isolating” or
“stabilizing” enzyme substrate interactions that may be functionally too weak or have
multiple substrate targets
2. Enhance the catalytic activity of the enzyme through protein-protein interactions (allosteric
contribution). May involve sequential activation of enzymes bound to the scaffold
3. Colocalization of pathway components to a particular subcellular compartment. Also
referred to as an “anchoring role”. This anchoring role is thought to enhance the efficiency
of signal propagation for multi-enzyme pathways that can be activated by multiple
extracellular stimuli

79. Enzyme Compartmentation
1. Opposing pathways e.g. fat acid synthesis in the cytosol and oxidation in the mitochondrion.
2. Pathway may be partitioned away from its substrate- acetyl CoA should be transported from
the mitochondria to the cytoplasm for FA synthesis. FA should enter the mitochondria for
oxidation
3. SHUTTLE Mechanisms- Solves the problems related to translocation of metabolites across
membranes by are transformed into forms that can penetrate barriers.

80. Regulation by Isozymes (Isoenzymes)
• Multiple forms of enzyme that catalyze same reaction
• Different amino acid sequences (products of different genes)
• Expressed in different tissues or organelles, at different stages of development, to
meet different metabolic/regulatory criteria.
• Different kinetic parameters like Km, and/or different allosteric regulation, with
physiological consequences
• Example: hexokinase (in muscle) vs. glucokinase (in liver)
– muscle function = contraction; breaks down glucose for energy; gets glucose
from blood
– 1 major liver function = maintenance of blood [glucose] at ~4-5 mM; liver
takes up and stores excess glucose, or makes more glucose and exports it, as
needed
– Function of hexokinase/glucokinase: glucose entering cells from blood is
phosphorylated, trapping it inside (charged compound can’t get back out)
– Hexokinase I (muscle): low Km for glucose, ~0.1 mM (so working at ~Vmax
since cellular [glucose] ~2-5 mM); inhibited by product, glucose6-phosphate -- if
G-6-P is building up, muscle won’t take more in from blood.
– Hexokinase IV (Glucokinase) (liver): high Km for glucose, ~10 mM, so activity
regulated by blood [glucose]; not inhibited by product G-6-P.

81. Information from enzymes measurements in
serum
 Presence of disease
 Organs involved
 Aetiology /nature of disease: differential
diagnosis
 Extent of disease-more damaged cells-more
leaked enzymes in blood
 Time course of disease

82. Enzymes use in Assessment of cell damage and
proliferation
Changes in plasma enzyme levels may help to detect and localize
tissue cell damage or proliferation, or to monitor treatment and progress
of disease.
•Plasma enzyme levels depend on:
a) the rate of release from damaged cells which, in turn, depends on the rate at which
damage is occurring;
b) the extent of cell damage.
•In the absence of cell damage, the rate of release depends on:
a) the rate of cell proliferation;
b) the degree of induction of enzyme synthesis.
•These factors are balanced by :
a) the rate of enzyme clearance from the circulation (only partly known);

83. Enzyme markers of clinical significance
Enzyme (abbreviation) Clinical significance
Acid phosphatase (ACP) Prostatic carcinoma
Alkaline phosphatase (ALP) Obstructive liver diseases, bone disorders
Alanine transaminase (ALT,GPT) Hepatic disorders, viral hepatitis
Aspartate transaminase (AST,GOT) Myocardial infarction, hepatic disorders
Alpha-amylase (AMS) Acute pancreatitis
Aldolase (ALS) Skeletal muscle disorders
Creatine kinase (CK) Myocardial infarction, muscle disorders
Gamma-glutamyl transferase (GGT) Hepatic disorders
G-6-PD Drug-induced hemolytic anemia
Lactate dehydrogenase (LD) Myocardial infarction, hepatic disorders,
carcionomas
Lipase (LPS) Acute pancreatitis
Leucine aminopeptidase (LAP) Hepatobiliary disorders
5’-Nucleotidase (5’NT) Hepatobiliary disorders
Pseudocholineesterase (PChE) Organophosphate poisoning
(butyrylcholine as substrate)
Ceruloplasmin (Copper-oxidase) Wilson’s disease (abnormal Cu metabolism)

84. Half-lives of clinically important enzymes in
plasma
Table 1. Half-lives of clinically important enzymes in plasma
Enzyme Range (hours)
Lactate dehydrogenase (LD)
LD-1 (H4) 50-70
LD-5 (M4) 8-14
Alanine transaminase (ALT, GPT) 40-50
Aspartate transaminase (AST, GOT)
mitochondrial AST 6-7
cytosolic AST 12-17
Creatine kinase (CK)
CK-MM 10-20
CK-MB 7-17
CK-BB 3
Alkaline phosphatase (ALP)
liver ALP 190-230
bone ALP 30-50

85. Serum normal (reference) ranges of clinical
enzymes
Enzyme Abbreviation Range Stability
(male> female)
Acid phosphatase ACP, AP 0.2-5.0 U/L +
Alkaline phosphatase ALP 30-95 U/L +++
Alanine transaminase ALT, G PT 6-37 U/L ++++
Aspartate transaminase AST, GOT 5-30 U/L +++
Alpha-amylase AMS 95-290 U/L ++++
Aldolase ALS 1.5-8.0 U/L ++++
Creatine kinase CK, CPK 15-160 U/L --
Gamma-glutamyl transferase GGT 6-45 U/L ++++
Glucose-6-phosphate dehydrogenase G-6-PD 0-0.2 U/L +++
Lactate dehydrogenase LD, LDH 100-225 U/L +
Lipase LPS 0-2 U/ml ++++
Leucine aminopeptidase LAP 11-30 U/L +++
5’-Nucleotidase 5’NT 2-15 U/L +++
Pseudocholineesterase PChE 5-12 U/ml ++++
Ceruloplasmin (Copper-oxidase) 0.2-0.6 g/L

86. Plasma enzyme patterns in disease: diagnosis &
monitor
Time sequence of changes in plasma enzymes after myocardial infarction (hours,
h; days, d)
Enzyme Onset of Peak activity Degree of Duration of
elevation (h) (h) elevation elevation (d)
CK (total) 4-8 12-24 5-10 x normal 3-4
CK-MB 4-8 24-36 5-15 x normal 2-3
AST (GOT) 8-12 24-48 2-3 x normal 4-6
LD 12-24 48-72 2-3 x normal 10
LD-1>LD-2 12-24 5
Remarks: Precision of diagnosis can be improved by
• estimations of more than one enzyme
• isozyme determinations
• serial enzyme estimations

87. LEVELS OF ENZYMES IN DISEASES
INVOLVING LIVER DAMAGE
In viral hepatitis
Rapid rise in
transaminases (AST &
ALT) in serum occurs
even before bilirubin
rise is seen

88. LEVELS OF ENZYMES IN MYOCARDIAL
INFARCTION
CK
CK-MB
AST
LDH
HBDH
AST and CK rise in 6
hours following acute
myocardial infarction
HBDH and LDH are
elevated much later and
remains high for a
longer period of days

89. Enzymes as therapeutic agents, drugs
Enzymes used in therapy are genetically engineered proteins.
Enzyme Disease/therapy
Protease, e.g., Streptokinase, Clot lysis in myocardial
Activase(plasminogen activator) infarction, trauma, bleedings
Aspariginase, Acute lymphocytic leukemia
e g., Oncospar (pepasparagase)
Adenosine deaminase, Severe combined immuno-
e.g., Adagen deficiency syndrome (SCID)
Superoxide dismutase, Head injury (clinical trial phase)
e.g., Dismutec peg
Nanoenzyme/nanozyme (2007)-catalase Parkinson’s disease- (attenuate
neuroinflammatory process)

90. Enzymes as drug targets
Enzyme targeting Drug
Dihydrofolate reductase Antifolates: methrotrexate (cancer),
(Folate metabolism) pyrimethamine (protozoa, malaria)
trimethoprim (bacteria)
Xanthine oxidase Allopurinol (hyperuricemia, gout)
(Purine metabolism)
Thymidylate synthase 5-Fluorouracil &
(Pyrimidine metabolism) 5-fluorodeoxyuridine (cancer)
Glycopeptide transpeptidase Antibiotics, penicillin
HIV-Reverse transcriptase 3’-azido-2’,3’-dideoxythymidine (AZT)
HIV & SARS proteases Ritonavir, saquinavir (clinical trial phase)

91. Enzymes abnormalities in metabolisms
1. Excess enzyme activity
Gout is characterized by elevated uric acid
levels in blood and urine, due to
overproduction of de novo purine
nucleotides.
E.g., Excess PRPP synthase activity (X-
linked recessive inheritance pattern)
purine nucleotides
then leads to increase degradation of purines
to uric acid through xanthine oxidase.

92. Enzymes abnormalities in metabolisms
2. Enzyme deficiency
Identification and treatment of enzyme deficiency.
Enzyme deficiencies usually lead to increased accumulation of specific metabolites
in plasma and hence in urine. This is useful in pinpointing enzyme defects.
E.g., De novo pyrimidine pathway: defects of OPRT and OMPDC leads
to accumulation of orotate ----> Hereditary orotic aciduria
(Gene mapping, 3q13; inheritance pattern, autosomal recessive).

93. Enzymes abnormalities in metabolisms
3. Enzyme defects found in all human metabolisms.
Enzyme defect Disease Metabolism/molecule involved
Pyruvate kinase Deficiency/Anemia Glycolysis
Pyruvate dehydrogenase Pyruvate/Krebs cycle
Chronic lactic acidosis
G-6-PD Deficiency Pentose phosphate pathway
Glycogen debranching enzyme
Cori (type III ) Gylcogen storage
Iduronate sulfatase Hunter Mucopolysaccharides
Acyl-CoA dehydrogenase
Deficiency Fatty acid oxidation
Hexoaminidase A Tay-sachs Lipid/sphingolipid storage
Acid lipase Deficiency Cholesterol/Triacylglycerol (TAG)
HGPRT Lesch-Nyhan Purine
OPRT/OMPDC Orotic aciduria Pyrimidine
Phenylalanine hydroxylase Amino acids/Phe
Phenylketonuria
Arginase Deficiency Amino acids/Arg /Urea cycle
Lysyl hydroxylase Ehlers-Danlos Collagen
Examples of enzyme defects.

94. 94
Enzyme Mechanisms
• In this section we will study the reaction
mechanisms for some specific enzyme-
catalyzed reactions:
– Proteases (Zymogens):
• Chymotrypsin, trypsin, elastase (nucleophillic attack)

95. Proteases classification and mechanisms
1) Cysteine Proteases
2) Aspartyl Proteases
3) Metalloproteases
4) Serine Proteases
Proteases - Enzymes that specifically cut other proteins and are important in regulation

96. 1) Cysteine Proteases
MECHANISM: In cysteine proteinases, catalysis proceeds
through the formation of a covalent intermediate and
involves a cysteine and a histidine residue. The essential
Cys and His play the same role as Ser and His respectively
in Serine proteases as discussed later. The nucleophile is a
thiolate ion that is stabilized through the formation of an ion
pair with neighbouring imidazolium group of His. The
attacking nucleophile is the thiolate-imidazolium ion pair in
both steps.
EXAMPLES:
Medically interesting cysteine proteases include:
- mammalian enzymes such as cathepsins B and L, which are involved in cancer
growth and metastasis, and cathepsin K, which is important for bone degradation
an osteoporosis.
- Cruzipain and cruzain from Trypanosoma cruzi, which cause Chagas' disease,
a permanent infection that affects more than 25 million people annually in South
America and causes more than 45,000 deaths per year, and falcipain, from
Plasmodium falciparum, which causes malaria.
- Caspases, which are key mediators of apoptosis.

97. 2) Aspartyl Proteases
MECHANISM: In contrast to cysteine (and serine)
proteases, catalysis by aspartic proteinases do not involve
a covalent intermediate, even though a tetrahedral
intermediate is transiently formed. Rather, nucleophilic
attack is achieved by two simultaneous proton transfers:
one from a water molecule to one of the two carboxyl
groups and a second one from the carbonyl oxygen of the
substrate with the concurrent CO-NH bond cleavage. This
general acid-base catalysis, which may be called a "push-
pull" mechanism leads to the formation of a non covalent
neutral tetrahedral intermediate
EXAMPLES:
Plasmepsin, which is produced in the parasite that causes malaria, is part of a
closely related group of enzymes known as aspartyl proteases. Plasmepsin is believed
to play a key role in the digestion of the human host's hemoglobin, the major nutrient
source for the parasite.
HIV protease permits viral maturation.
BACE, an aspartyl protease involved in the amyloid peptide generation of Alzheimer's
disease.

98. 3) Metalloproteases (Zn)
MECHANISM: Many enzymes contain the sequence HEXXH,
which provides two histidine ligands for binding of zinc. A third
Zn ligand is either a glutamic acid (thermolysin, neprilysin,
alanyl aminopeptidase) or a histidine (astacin, serralysin).
Other families exhibit a distinct mode of binding of a Zn ion. The
catalytic mechanism involves formation of a non covalent
tetrahedral intermediate after the attack of a zinc-bound water
molecule on the carbonyl group of the scissile bond. This
intermediate can be further decomposed by transfer of the
glutamic acid proton to the leaving group.
EXAMPLES:
Matrix metalloproteinases (MMPs) are a family of enzymes that are responsible for the
degradation of extracellular matrix components such as collagen, laminin and
proteoglycans. These enzymes are involved in normal physiological processes such as
embryogenesis and tissue remodeling and may play an important role in arthritis,
periodontitis, and metastasis.
ACE is a metalloprotease that catalyses the conversion of angiotensin I into angiotensin
II, which leads to vasoconstriction. ACE inhibitors, were originally used as
antihypertensives, but have significantly improved the treatment of other cardiovascular
diseases and are now used to treat heart failure and even prevent heart attacks in at-risk

99. 4) Serine Proteases
MECHANISM:
The key active site groups are Ser, His and Asp. These groups are in the same orientation in all
the serine proteases. Their roles are basically as follows: the imidazole (His) acts as a general
base-general acid, first to activate the serine OH for nucleophilic catalysis, then the leaving
group (by general acid cat.), then as a general base it activates water to attack the covalent
acyl-enzyme intermediate. The Asp serves to orient the His side chain and to provide an
appropriate electrostatic environment.
EXAMPLES:
• Trypsin and Chymotrypsin are digestive enzymes in the small intestine
• Subtilisin is a bacterial serine protease that is used in laundry detergents
• TADG-14 is a novel extracellular serine protease that has been identified and cloned from
ovarian carcinoma. It is uniquely expressed in ovarian cancer, both in early stage and overt
carcinomas. It is seldom or not at all expressed in normal adult tissues and has not been
detected in other fetal tissues. It offers the potential as a target for therapeutic intervention
through down-regulation of its protease activity.
• NS3/4A is a serine protease in Hepatitis C (HCV) that is important in viral maturation.
• Factor VIIa, Factor Xa, and thrombin are serine proteases in blood coagulation pathway

100. Trypsin and Chymotrypsin are very well studied serine proteases whose
structures and mechanisms are well understood
They catalyze the hydrolysis of internal peptide bonds (thus an endoprotease). Trypsin
cleaves on the carboxyl side of basic side chains (Lys, Arg), whereas
chymotrypsin cleaves on the carboxyl side of aromatics (Phe, Tyr, Trp)
The active site consists of a “catalytic triad”:
1) Serine, to which the substrate binds
2) Histidine, which has the ability to donate and accept protons.
3) Aspartate, which has the ability to accept protons.
These residues are polar (hydrophilic) so would not ordinarily be
found on the "interior” of a protein.
Though they are in close proximity in the 3D structure, they are not adjacent in the
primary sequence (Ser-195, His-57, Asp-102).
102

101. First stage in peptide bond hydrolysis: acylation. Hydrolysis of the peptide bond
starts with an attack by the oxygen atom of the Ser195 hydroxyl group on the carbonyl
carbon atom of the susceptible bond. The carbon-oxygen bond of this carbonyl group
becomes a single bond, and the oxygen atom acquires a net negative charge. The
four atoms now bonded to the carbonyl carbon are arranged as a tetrahedron.
Transfer of a proton from Ser195 to His57 is facilitated by Asp102 which (i) precisely
orients the imidazole ring of His57 and (ii) partly neutralizes the positive charge that
develops on His57 during the transition state. The proton held by the protonated form
of His57 is then donated to the nitrogen atom of the peptide bond that is cleaved. At
this stage, the amine component is hydrogen bonded to His57, and the acid
component of the substrate is esterified to Ser195. The amine component diffuses
away.
Oxyanion
hole

102. Second stage in peptide hydrolysis: deacylation. The acyl-enzyme
intermediate is hydrolyzed by water. Deacylation is essentially the
reverse of acylation with water playing the role as the attacking
nucleophile, similar to Ser195 in the first step. First, a proton is drawn
away from water. The resulting OH- attacks the carbonyl carbon of the
acyl group that is attached to Ser195. As in acylation, a transient
tetrahedral intermediate is formed. His57 then donates a proton to the
oxygen atom of Ser195, which then releases the acid component of the
substrate, completing the reaction.
Oxyanion
hole

103. (From “Mark’s Basic Medical Biochemistry – A clinical
approach”)

104. How is specificity obtained among trypsin-like serine proteases?

105. Specificity Difference of Chymotrypsin, Trypsin, and
Elastase
nonpolar pocket Asp (negatively charged)
vs. Ser in Chymotrypsin
no pocket present
as two Gly in chymotrypsin
are replaced by Val and Th
• Substrate specificity
– Chymotrypsin: aromatic or bulky nonpolar side
chain
– Trypsin: Lys or Arg
– Elastase: smaller & uncharged side chains
• Small structural difference in the binding site explains
the substrate specificity

106. Serine Protease Family
Chymotrypsin & elastase
main chain conformation
(superimposed)
• Serine Proteases
– Chymotrypsin
– Trypsin
– Elastase
• Similarity
– Similar 3D structure
– Catalytic triad
– Oxyanion hole
– Covalent acyl-enzyme intermediate
– Secreted by pancrease as inactive precursors

107. 107
Enzyme Mechanisms –
Pancreatic Trypsin Inhibitor
• Another way in which the body is
protected from undesirable
proteolytic action is to synthesize
competitive inhibitors, such as the
pancreatic trypsin inhibitor (~6kD).
When bound, this inhibitor turns the critically important histidine
in the charge relay network out of its normal plane, breaking up
the smooth flow of electrons across the amino acid triad. This
greatly reduces the ability of serine to form an alkoxide,
impeding the initial step in the enzyme mechanism. Upon
dilution in the duodenum, the inhibitor dissociates, freeing the
enzyme for action.

108. 108
Enzyme Mechanisms – Elastase Inhibitor
An similar important inhibitor of a different zymogen,
elastase, is the 53-kD protein α1-antitrypsin. (“anti-
elastase” would be a better name.)
This inhibitor binds to elastase in the lungs, helping
prevent proteolytic damage to the alveolar linings
caused by elastase.
A “type Z” mutation substitutes lys for glu-53, resulting in
compromised secretion from liver cells where it is
synthesized. The resulting decreased level of this
inhibitor in the lungs leads to emphysema.

109. 109
Enzyme Mechanisms – Elastase Inhibitor
• Smoking also damages this α1-antitrypsin inhibitor. Smoke
oxidizes methionine-358, a residue essential for binding to
elastase. The reduced affinity of elastase for the α1-antitrypsin
inhibitor frees the enzyme to destroy tissues in the lung.

110. Thank you