by SUNIL SHAH (BOND KING)

2. Enzymes are protein catalysts responsible
for most of the chemical reactions of the
body
They are found in: cells and all tissues;
serum to which gain access from injured
cells, cells undergone stress

3. in disease states, caused in increased
membrane permeability
caused by increase rates of
intracellular synthesis
subsequent diffusion of enzymes
Enzymes found in serum will help physicians
diagnose certain disease and aid in the
monitoring of the disease condition

4. Increase serum levels
increased release of enzyme from
source
o necrosis
o increased membrane permeability
increased size of tissue source of
enzyme
impaired excretion of enzyme
increased enzyme synthesis

5. Decreased serum levels
decreased formation
- Genetic
- Acquired
- Enzyme inhibition
poisoning
lack of cofactors

6. Alkaline phosphatase – diagnose
osseous and hepatobiliary diseases
Acid phosphatase – in the diagnosis of
prostate cancers
Amylase and lipase – diagnosis of
pancreatic disease
SGPT – liver diseases (hepatitis)
SGOT – cardiac and liver diseases

7. Catalyst
A substance that enhances the rate of a
chemical reaction but is not permanently
altered by the reaction
Decreases the activation of energy required for a
chemical reaction and provides an alternative
reaction pathway that requires less energy

8. Enzymes are neither produced or
consumed in the reaction
Enzymes do not cause the reaction
but enhances the reaction to occur
Enzymes are long sequences of
amino acid

9. Enzymes are highly specific and
produce only the expected product
from given reactant or substrate
Enzymes acts on moderate pH and
temperature

10. Enzyme possess the following:
Active site – where the substrate fits
before converted to corresponding
product
Allosteric site – which binds effector
molecules or modifiers which regulates
enzyme activity

11. Apoenzyme – are protein portion of
the enzyme
Coenzyme – non-protein component;
main participant during reaction with a
substrate

12. help in enzymatic activity by orienting
properly the substrate with the active site.
Coenzyme – organic cofactor: NADH,
NADPH, FAD and FMN
Activator - usually metallic ions tightly
bound to enzymes: Mg, Zn++, Cu++

13. Other enzymes are pro-enzymes or
zymogens that are inactive but once
released to their target sites they become
activated.
Isoenzymes – enzymes with similar
enzymatic activities but differ in their
chemical structure and origins

14. They are unchanged during the
course and termination of
chemical reaction.
They demonstrate great
specificity:
Absolute – pyruvate kinase
Group - phosphatases
Bond - hydrolases
Stereospecificity - transaminases

15. Enzyme binds to a single type of
substrate because of
complementary structures of the
active site and substrate
Substrate’s overall shape and
charge allows to interact with
enzymes active site

16. Flexibility structure of protein is
taken into account
Substrate does not precisely fit
into the rigid active site instead
a non-covalent interaction of
enzyme-substrate change it
thus conforming to the shape of
active site to the shape of the
substrate

17. Substrate Concentration
Michaelis-Menten hypothesis: the rate of
substrate conversion to product is
determined by
 substrate concentration and
 rate of dissociation of enzyme substrate complex
first-order kinetics
 At constant enzyme concentration, the velocity or speed
of the enzyme reaction initially increases as the substrate
concentration increases.
Reaction rate is dependent on the substrate
concentration

18. Zero-order kinetics
The point at which the further addition of
substrate does not anymore change the velocity
of enzyme reaction.
The reaction is now dependent on the
enzyme concentration
Km
Represents the substrate concentration where
the velocity is ½ the maximum
Represents the substrate concentration at which
the enzyme yields half the possible maximum
velocity
It is also the measure of affinity of the enzymes
with its substrate

19. Enzyme concentration
The higher the enzyme concentration the
higher is the reaction rate. (true only in
zero-order kinetics)
pH
optimal pH varies with each enzyme

20. Temperature
most denatures at 60oC
usually optimum at 37oC
there is a characteristic increase in the
reaction rate for every 10oC increase
before denaturation

21. Cofactors
metals – transition metals (Zn++. Cu++ and
Fe++) – effective electophiles
Inhibitors
Competitive (compete for active site)
Non-competitive (binds with enzyme at
place other than active site)
Uncompetitive (binds with E-S complex)

22. Enzymes expressed in units that represent
one of the following:
Increased concentration of one of the
products  substrate and coenzyme
The rate of change of any 3 unit is a measure
of rate of reaction
The catalytic rate is proportional to its
concentration at normal condition

23. MICHEALIS-MENTEN HYPOTHESIS
k1 k3
E + S  (ES)  E + P

k2
where: k1 is rate constant for ES formation
k2 is rate constant for ES dissociation
k3 is rate constant for product
formation and release from active
site

24. International Union of Biochemistry (IUB)
Classify and name enzymes according to the type of
chemical reaction it catalyzes
Using the name of the substrate or group on which
the enzymes acts and added by suffix “-ase”
Examples:
 Urease  hydrolyzes urea
 Amylase  metabolizes starch/amylum
 Phosphatase  acting on phosphate esters

25. Biochemical functions, indicating
substrate, class of reaction catalyzed
designated by individual identification
numbers
For clarity, the raction is also identified
(examples are carbonic anhydrase, D-
amino acid oxidase and succinic
dehydrogenase)

26. Systemic name – nature of the reaction catalyzed
is associated with unique numercal code
designation
Of two parts: substrate(s) acted upon and a word
ending with –ase indicating the reaction involved
Example: L-Lactate:NAD+ oxidoreductase
Trivial or practical name – may be identical to the
systemic name but is often a simplification of it –
useful in everyday work

27. Example:
EC 1.1.1.27  L-Lactate:NAD+ oxidoreductase 
lactate dehydrogenase
EC denotes Enzyme Commision
First number defines the class to which the enzyme
belongs

28. Assigned to 6 classes
1. oxidoreductases
2. transferases
3. hydrolaseses
4. lyases
5. isomerases
6. ligases

29. next two numbers indicate the subclass and sub-
subclass to which the enzyme is assigned
Example: may be differentiated from the amino
transferring subclass of the phosphate – transferring
category or the ethanol acceptor sub-class from that
accepting acyl group
The last number is the specific serial number given
to the enzyme within its subclass

30. 1. oxidoreductases - catalyzed oxidation-
reduction reactions
REDUCTION (addition of hydrogen to a double bond)
OXIDATION (removal of a hydrogen from a molecule to
leave a double bond)
The hydrogen is transferred with the use of coenzyme
L-lactate:NAD+ oxidoreductase catalyzed
pyruvate + NADH + H+  lactate + NAD+
subclasses: dehydrogenases, oxidases,
oxygenases, reductases, peroxidases, and
hydroxylases

31. 2. transferases – catalyzed reactions that
involve the transfer of groups from one
molecule to another (amine or phosphate
group)
ATP:creatine N-phosphotransferase (creatine kinase)
involves
ATP + creatine  ADP + creatine phosphate
trivial names include with prefix “trans”:
transcarboxylases, transmethylases and
transaminases

32. 3. hydrolases – catalyze reactions in which
the cleavage of bonds is accomplished by
adding water
Amylase (cleavage of –C-O-C- bonds in starch)
Lipase (breaks down triglycerides to form glycerol and free
fatty acids)
Acid phosphatase and alkaline phosphatase (remove
phosphate group from a variety of molecules)
subclasses: esterases, phosphatases, peptidases

33. 4. lyases – (lysis means “splitting”)
catalyze reactions in which groups are
removed to form a double bond or are
added to a double bond (C-C, C-S, and C-N
bonds)
Aldolase (EC 4.1.2.13, D-fructose-1,6-biphosphate-D-
glyceraldehyde-3-phosphate lyase) which cleaves the 6-
carbon molecule fructose-1,6-diphosphate to produce two
3-carbon compounds: glyceraldehyde-3-phosphate and
dihydroxyacetone phosphate
 subclasses: decarboxylases, hydratases, dehydratases,
deaminases, synthases

34. 5. isomerases – (heterogenous group)
catalyze several types of intramolecular
rearrangements
Where it involves in the conversion of one
isomer to another with examples of
transformation will include the change of cis
to trans; L-form to D-form; aldehyde to
ketone
the reactions are generally reversible

35. Triose phosphate isomerase (EC 5.3.1.1,
D-glyceraldehyde-3-phosphate ketol-
isomerase)
In the glycolytic pathway, involves in the
isomerization of glyceraldehyde-3-
phosphate (aldehyde) to dihydroxyacetone
phosphate (ketone)
epimerases – catalyze the inversion of
assymmetric carbon atoms
mutases – catalyze the intramolecular transfer of
functional group

36. 6. ligases – catalyze bond formation
between two substrate molecules forming a
larger molecule
Important in the activation of amino acids –
protein synthesis
Energy is always supplied by ATP
Aminoacyl-tRNA

37. 1. Substrate Measurement
 This starts with a high substrate
concentration
1. Product Measurement
 This starts with zero initial product level
 This is more accurate

38. Endpoint analysis
the reaction is initiated by addition of substrate
and is allowed to proceed for a period of time
measurement of substrate or product is done at
the end of the reaction
Multipoint assay
this measures the change in the concentration of
the indicator substance at several intervals during
the course of the assay

39. Kinetic assay
this involves measurement of change in
concentration as a function of time
closely monitored at short interval
has advantage over the end point
if the concentration of the substrate is sufficiently
high in comparison to enzyme then rate of
reaction will be proportional to the concentration
of enzyme
thus the amount of product formed in a given
period of time would be proportional to the
amount of active enzyme present, with all other
factors remaining constant

40. Use of coupled reactions
enzymatic activity is measured by
coupling the activity with colorimetric
reaction
the colored product is measured
spectrophotometrically

41. 1. HEMOLYSIS
 May cause falsely elevated enzyme concentration
1. ANTICOAGULANTS
 Many anticoagulants cause adverse effects on enzyme
inhibiton, therefore, serum is preferred over plasma
1. LACTESCENT OR MILKY SERUM
 May result in variable absorbance readings in
spectrophotometry

42. Enzymes are stable at 6oC for at least 24 hours and at
room temperature for lesser periods
For prolonged storage, use -20oC or lower
CK must be kept at -70oC
LD4 and LD5 – liver isoenzymes are inactivated at
refrigerator temperature

43. That’s all folks… Have a great day ahead!