1. MEDICAL BIOCHEMISTRY:
ENZYMES
MR. GENARO F. ALDERITE
JR,MSERM

2. ENZYMES
- A protein with catalytic properties due to its
power of specific activation

3. Characteristics of Enzymes
1) biological catalysts
2) not consumed during a chemical reaction
3) speed up reactions from 1000 - 1017, with a mean
increase in rate of 00,000
4) exhibit stereospecificity --> act on a single
stereoisomer of a substrate
5) exhibit reaction specificity --> no waste or side
reactions

4. Classification of Enzyme
Specificity
a. Absolute specificity: substrate
Succinic dehydrogenase- succinic acid to fumaric
acid
b. Linkage specificity:reaction that break bonds
Thrombin- acids arginine and glycine
c. Reaction specificity: reactions
Esterases- hydrolysis of esters
d. Group Specificity: compounds
chymotrypsin- catalyzes only protein that contains
phenylalanine, tryptophan and tyrosine

5. Classification of Enzymes:
1. According to its composition:
a. Simple enzymes-
b. Complex enzymes
holoenzyme - a complete, catalytically
active enzyme including all co-factors
apoenzyme - the protein portion of a
holoenzyme minus the co-factors
prosthetic group - a metal or other co-
enzyme covalently bound to an

6. 2. Class of organic chemical reaction catalyzed:
a. Oxidoreductase - catalyze redox reactions
*dehydrogenases, oxidases, peroxidases, reductases
Dehydrogenase-catalyze the removal of H from
a substrate
Oxidases- activate oxygen so that it will readily c
ombine with a substrate
b. Transferases - catalyze group transfer reactions;
often require coenzymes

7. c. Hydrolases - catalyze hydrolysis reactions
Carbohydrates
1. ptyalin- salivary amylase
-catalyze the hydrolysis of starch to dextrin
and maltose
2. sucrase- hydrolysis of sucrose to glucosE and
fructose
- intestinal juices
3. maltase- hydrolysis of maltose to glucose
4. Lactase- hydrolysis of lactose to glucose and
galactose

8. 5. amylopsin- pancreatic amylase
- hydrolysis of starch to dextrins and
maltose *from pancreas to
Sintestine*
Esters- catalyze the hydrolysis of esters into acids
and alcohol
1. Gastric lipase- hydrolysis of fats to fatty
acids and alcohol
- part of the gastric juices
2. Steapsin- ( pancreatic lipase)
- hydrolysis of fats to fatty acids and

9. Proteases- catalyze the hydrolysis of derived
proteins and amino acids
1. pepsin- hydrolysis of protein to
polypeptides
2. trypsin- found in pancreatic
juice
3. chymotrypsin

10. Hydrolysis Reaction
β-galactosidase
Lactose + H2O Glucose + Galactose

11. d. Lyases - lysis of substrate; produce contains
double bond
e. Isomerases - catalyze structural changes;
isomerization
f. Ligases - ligation or joining of two substrates
with input of energy, usually from ATP
hydrolysis; often called synthetases or
synthases

12. Chemical reactions
• Chemical reactions need an initial input of
energy = THE ACTIVATION ENERGY
• During this part of the reaction the
molecules are said to be in a
TRANSITION STATE

13. Reaction Pathway

15. Making Reactions Go Faster
• Increasing the temperature make molecules move
faster
• Biological systems are very sensitive to temperature
changes.
• Enzymes can increase the rate of reactions without
increasing the temperature.
• They do this by lowering the activation energy.
• They create a new reaction pathway “a short cut”

16. An Enzyme Controlled
Pathway

18. ENZYMATIC REACTION
PRINCIPLES
• Biochemically, enzymes are highly specific for their
substrates and generally catalyze only one type of
reaction at rates thousands and millions times higher
than non-enzymatic reactions. Two main principles
to remember about enzymes are 1) they act as
CATALYSTS (they are not consumed in a reaction
and are regenerated to their starting state) and 2)
they INCREASE THE RATE of a reaction
towards equilibrium (ratio of substrate to product),
but they do not determine the overall equilibrium of
a reaction.

19. CATALYSTS
• A catalyst is unaltered during the course of a
reaction and functions in both the forward and
reverse directions. In a chemical reaction, a catalyst
increases the rate at which the reaction reaches
equilibrium, though it does not change the
equilibrium ratio. For a reaction to proceed from
starting material to product, the chemical
transformations of bond-making and bond-breaking
require a minimal threshold amount of energy,
termed activation energy. Generally, a catalyst
serves to lower the activation energy of a particular
reaction.

20. ENZYMATIC REACTION
PRINCIPLES (cont)
• The energy maxima at which the reaction has the
potential for going in either direction is termed the
transition state. In enzyme catalyzed reactions, the
same chemical principles of activation energy and
the free energy changes (∆Go) associated with
catalysts can be applied. Recall that an overall
negative ∆Go indicates a favorable reaction
equilibrium for product formation. As shown in an
enzyme catalyzed reaction, and in the graph, the net
effect of the enzyme is to lower the activation
energy required for product formation.

21. Binding Energy
• The graph of activation energy and free energy changes
for an enzymatic reaction also indicates the role binding
energy plays in the overall process. Due to the high
specificity most enzymes have for a particular substrate,
the binding of the substrate to the enzyme through
weak, non-covalent interactions is energetically
favorable and is termed binding energy. The same
forces important in stabilizing protein conformation
(hydrogen bonding and hydrophobic, ionic and van der
Waals interactions) are also involved in the stable
binding of a substrate to an enzyme.

22. Reaction Rates
• The rate of the reaction is determined by several factors
including:
A. The concentration of substrate
B. Temperature
C. pH.

23. Effect of Temperature
A reaction rate will generally
increase with increasing
Temperature due to increased
kinetic energy in the system until
a maximal velocity is reached.
Above this maximum, the kinetic
energy of the system exceeds the
energy barrier for breaking weak
H-bonds and hydrophobic
interactions, thus leading to
unfolding and denaturation of the
enzyme and a decrease in reaction
rate.

24. Q10 (the temperature coefficient) = the increase in
reaction rate with a 10°C rise in temperature.
For chemical reactions the Q10 = 2 to 3
(the rate of the reaction doubles or triples with every
10°C rise in temperature)
Enzyme-controlled reactions follow this rule as they
are chemical reactions
BUT at high temperatures proteins denature
The optimum temperature for an enzyme controlled
reaction will be a balance between the Q10 and
denaturation.

25. The effect of temperature
Q10 Denaturation
Enzyme
activity
0 10 20 30 40 50
Temperature / °C

26. The effect of temperature
For most enzymes the optimum temperature is
about 30°C
Many are a lot lower, cold water fish will
die at 30°C because their enzymes denature
A few bacteria have enzymes that can withstand
very high temperatures up to 100°C
Most enzymes however are fully denatured at 70°C

27. Effect of pH
Variations in pH can affect a
particular enzyme in many ways,
especially if ionizable amino acid
side chains are involved in binding
of the substrate and/or catalysis.
Extremes of pH can also lead to
denaturation of an enzyme if the
ionization state of amino acid(s)
critical to correct folding are
altered. The effects of pH and
temperature will vary for different
enzymes and must be determined
experimentally.

28. Extreme pH levels will produce denaturation
The structure of the enzyme is changed
The active site is distorted and the substrate
molecules will no longer fit in it
At pH values slightly different from the enzyme’s
optimum value, small changes in the charges of the
enzyme and it’s substrate molecules will occur
This change in ionisation will affect the binding of
the substrate with the active site.

29. Optimum pH
values
Enzyme
activity Trypsin
Pepsin
1 3 5 7 9 11
pH

30. Theories on Enzyme Specificity
1. The Lock and Key Hypothesis
2. The Induced Fit Hypothesis

31. The Lock and Key Hypothesis
• Fit between the substrate and the active site of the
enzyme is exact
• Like a key fits into a lock very precisely
• The key is analogous to the enzyme and the
substrate analogous to the lock.
• Temporary structure called the enzyme-substrate
complex formed
• Products have a different shape from the substrate
• Once formed, they are released from the active site
• Leaving it free to become attached to another
substrate

32. S
E
E
E
Enzyme may be
used again
Enzyme-substrate P
complex
P
Reaction coordinate

33. The Induced Fit Hypothesis
• Some proteins can change their shape
(conformation)
• When a substrate combines with an enzyme, it
induces a change in the enzyme’s conformation
• The active site is then moulded into a precise
conformation
• Making the chemical environment suitable for the
reaction
• The bonds of the substrate are stretched to make
the reaction easier (lowers activation energy)

34. The Induced Fit Hypothesis
Hexokinase (a) without (b) with glucose substrate
http://www.biochem.arizona.edu/classes/bioc462/462a/NOTES/ENZYMES/enzyme_mechanism.html

35. Hexokinase Active Site:
Glucose vs. Galactose Binding

36. LOCK-AND-
KEY
INDUCED FIT

37. Catalytic Mechanisms: Types
• Four types of catalytic mechanisms will be
discussed:
• binding energy catalysis
• general acid-base catalysis
• covalent catalysis
• metal ion catalysis

38. Acid-Base
Catalysis
Many reactions involve the formation of normally unstable, charged
intermediates. These intermediates can be transiently stabilized in an
enzyme active site by interaction of amino acid residues acting as weak
acids (proton donors) or weak bases (proton acceptors). The general
acid and general base forms of the most common and best characterized
amino acids involved in these reactions are shown above.

39. Acid-Base Catalysis (cont)
• The preceding functional groups can potentially serve
as either proton donors or proton acceptors. This is
dependent on many factors including the molecular
nature of the substrate, any co-factors involved, and the
pH of the active site (which would determine the
ionization state of an amino acid side chain). For acid-
base catalysis, histidine is the most versatile amino acid
due to its pKa which means that in most physiological
situations it can act as either a proton donor or proton
acceptor. Generally these amino acids will interact
together with the substrate, or in conjunction with
water or other weak, organic acids and bases found in
cells.

40. Binding Energy Catalysis
• Binding energy accounts for the overall lowering of
activation energy for a reaction, and it can also be
considered as a catalytic mechanism for a reaction. Several
catalytic factors in the binding of a substrate and enzyme
can be considered: 1) transient limiting of substrate and
enzyme movement by reducing the relative motion (or
entropy) of the two molecules, 2) solvation disruption of the
water shell is thermodynamically favorable, and 3) substrate
and enzyme conformational changes. All three of these
factors individually or in combination are utilized to some
degree by an enzyme. While in some instances these forces
alone can account for catalysis, they are frequently
components of a complex catalytic process involving
factors discussed for the other types of catalytic

41. Covalent Catalysis
• This mechanism involves the transient
covalent binding of the substrate to an
amino acid residue in the active site.
Generally this is to the hydroxyl group of a
serine, although the side chains of
threonine, cysteine, histidine, arginine and
lysine can also be involved.

42. Metal Ion Catalysis
• Various metals, all positively charged and
including zinc, iron, magnesium, manganese
and copper, are known to form complexes with
different enzymes or substrates. This metal-
substrate-enzyme complex can aid in the
orientation of the substrate in the active site,
and metals are known to mediate oxidation-
reduction reactions by reversible changes in
their oxidation states (like Fe3+ to Fe2+).

43. Summary of Catalytic
Mechanisms
• In general, more than one type of catalytic
mechanism will occur for a particular enzyme
via various combinations of binding energy,
acid-base, covalent and metal catalysis.
Enzymes as a whole are incredibly diverse in
their structures and the types of reactions that
they catalyze, therefore there is also a large
diversity of catalytic mechanisms utilized, the
basis of which must be determined
experimentally.

45. Inhibitors
• Inhibitors are chemicals that reduce the rate of
enzymic reactions.
• The are usually specific and they work at low
concentrations.
• They block the enzyme but they do not usually
destroy it.
• Many drugs and poisons are inhibitors of
enzymes in the nervous system.

46. The effect of enzyme inhibition
• Irreversible inhibitors: Combine with the
functional groups of the amino acids in the active
site, irreversibly.
Examples: nerve gases and pesticides, containing
organophosphorus, combine with serine residues
in the enzyme acetylcholine esterase.

47. Reversible inhibitors: These can be washed out of
the solution of enzyme by dialysis.
Two Categories:
Competitive: These compete with the substrate
molecules for the active site.
The inhibitor’s action is proportional to its
concentration.
Resembles the substrate’s structure closely.

48.  Non-competitive: These are not influenced by
the concentration of the substrate. It inhibits by
binding irreversibly to the enzyme but not at
the active site.
Examples
• Cyanide combines with the Iron in the enzymes
cytochrome oxidase.
• Heavy metals, Ag or Hg, combine with –SH
groups.
T hese can be removed by using a chelating agent
such as EDTA.

49. Medicine inhibitors:
a. Methotrexate in cancer chemotherapy to semi-
selectively inhibit DNA synthesis of malignant
cells
b. Aspirin to inhibit the synthesis of
prostaglandins which are at least partly
responsible for the aches and pains of arthritis
c. Sulfa drugs to inhibit the folic acid synthesis
that is essential for the metabolism and growth of
disease-causing bacteria

50. Activators: are molecules that
increase activity.
Examples:
Lipases- Used to assist in the removal of fatty
and oily stains.
Amylases Detergents- for machine dish washing
to remove resistant starch residues.
Papaine- To soften meat for cooking.

51. Clinical Use of Enzymes
• Enzyme Activity in Body Fluids Reflects Organ
Status:
• Cells die and release intracellular contents;
increased serum activity of an enzyme can be
correlated with quantity or severity of damaged
tissues (ex. creatine kinase levels following heart
attack)
• Increased enzyme synthesis can be induced and
release in serum correlates with degree of
stimulation (ex. alkaline phosphatase activity as a
liver status marker)

52. Clinical Use of Enzymes (cont)
• Enzyme Activity Reflects the Presence of
Inhibitors or Activators
• Activity of serum enzymes decreases in presence
of an inhibitor (ex. some insecticides inhibit serum
cholinesterases)
• Determine co-factor deficiencies (like an essential
vitamin) by enzyme activity (ex. add back vitamin
to assay, if activity increases, suggests deficiency
in that vitamin)

53. Clinical Use of Enzymes (cont)
• Enzyme activity can be altered genetically
• A mutation in an enzyme can alter its substrate
affinity, co-factor binding stability etc. which can be
used as a diagnostic in comparison with normal
enzyme
• Loss of enzyme presence due to genetic mutation as
detected by increased enzyme substrate and/or lack
of product leading to a dysfunction
• NOTE: PCR techniques that identify specific
messenger RNA or DNA sequences are replacing
many traditional enzymatic based markers of
genetic disease

54. Enzymes in the Diagnosis of
Pathology
The measurement of the serum levels of
numerous enzymes has been shown to be of
diagnostic significance. This is because the
presence of these enzymes in the serum
indicates that tissue or cellular damage has
occurred resulting in the release of
intracellular components into the blood .

55. Commonly assayed enzymes :
a.amino transferases:
b. alanine transaminase, ALT (sometimes still
referred to as serum glutamate-pyruvate
aminotransferase, SGPT)
c. aspartate aminotransferase, AST (also referred to
as serum glutamate-oxaloacetate aminotransferase,
SGOT);
d. lactate dehydrogenase, LDH;
e. creatine kinase, CK (also called creatine
phosphokinase, CPK);
f. gamma-glutamyl transpeptidase, GGT.

56. -The typical liver enzymes measured are AST
(aspartate aminotransferase), and
ALT(Alanine transaminase) .
-Normally in liver disease or damage that is
not of viral origin the ratio of ALT/AST is less
than 1. However, with viral hepatitis the ALT/
AST ratio will be greater than 1.

57. The 5 types and their normal distribution and levels in
non-disease/injury are listed below. (lactate
dehydrogenase )
• LDH 1 – Found in heart and red-blood cells and is
17% – 27% of the normal serum total.
• LDH 2 – Found in heart and red-blood cells and is
27% – 37% of the normal serum total.
• LDH 3 – Found in a variety of organs and is 18% –
25% of the normal serum total.
• LDH 4 – Found in a variety of organs and is 3% –
8% of the normal serum total.
• LDH 5 – Found in liver and skeletal muscle and is
0% – 5% of the normal serum total.

58. • CPK( Creatine phosphokinase) is found primarily in
heart and skeletal muscle as well as the brain.
Therefore, measurement of serum CPK levels is a
good diagnostic for injury to these tissues. The levels
of CPK will rise within 6 hours of injury and peak by
around 18 hours. If the injury is not persistent the
level of CK returns to normal within 2–3 days. Like
LDH, there are tissue-specific isozymes of CPK and
there designations are described below.
• CPK3 (CPK-MM) is the predominant isozyme in
muscle and is 100% of the normal serum total.
• CPK2 (CPK-MB) accounts for about 35% of the
CPK activity in cardiac muscle, but less than 5% in
skeletal muscle and is 0% of the normal serum total.
• CPK1 (CPK-BB) is the characteristic isozyme in
brain and is in significant amounts in smooth muscle
and is 0% of the normal serum total.