Environmental factors such as temperature, pH, and concentrations of enzymes, substrates, and products can affect the rate of enzyme-catalyzed reactions. Enzyme activity is also regulated by cofactors, allosteric effectors, covalent modification, and induction/repression of enzyme synthesis. Enzymes exhibit varying degrees of specificity, from absolute specificity for a single substrate to bond or group specificity for classes of substrates. Isoenzymes are variants of the same enzyme found in different tissues.

1. factors Affecting Enzyme activity

2. 12
factors Affecting Enzyme activity
• Three factors:
1. Environmental Conditions
2. Activators-Cofactors and Coenzymes
3. Enzyme Inhibitors

3. Environmental FACTORS
Affecting Rate of Enzyme Reactions
 Temperature
 Hydrogen ion concentration(pH)
 Enzyme concentration
 Substrate concentration

4. EFFECT OF TEMPERATURE
 Raising the temperature increases the rate of enzyme
catalyzed reaction by increasing kinetic energy of reacting
molecules.
 Enzymes work maximum over a particular temperature known
as optimum temperature. Enzymes for humans generally
exhibit stability temperature up to 35-45 ᵒ C.
 The temperature coefficient is a factor Q₁₀ by which the rate of
biological processes increases for a 10 ᵒ C increase in
temperature.
 For most biological processes Q₁₀ = 2.
 However some times heat energy can also increase kinetic
energy to a point that exceed the energy barrier which results
in denaturing of enzymes.

5. RateofReaction
Temperature
40oC - denatures
5- 40oC
Increase inActivity
0 10 20 30 40 50 60
<5oC - inactive

6. EFFECT OF PH
 Rate of almost all enzymes catalyzed reactions depends on
pH
 Most enzymes exhibit optimal activity at pH value between 5
and 9
 High or low pH value than optimum value will cause ionization
of enzyme which result in denaturation of enzyme

7. Optimum pH for Selected Enzymes
• Most enzymes of the body have an optimum
pH of about 7.4
• However, in certain organs, enzymes operate
at lower and higher optimum pH values

8. PH AFFECTS THE FORMATION OF HYDROGEN BONDS
AND SULPHUR BRIDGES IN PROTEINS AND SO AFFECTS
SHAPE.
pepsin
trypsin arginase
2 4 8 106
pH
RateofReaction(M)
Acidic Basic

9. Enzyme Concentration and Reaction Rate
• The rate of reaction increases as enzyme concentration
increases (at constant substrate concentration)
• At higher enzyme concentrations, more enzymes are
available to catalyze the reaction (more reactions at
once).
• There is linear relationhip between reaction rate and
enzyme conc,

10. Enzyme Concentration and Reaction
Rate
• The rate of reaction increases as
enzyme concentration increases (at
constant substrate concentration)
• At higher enzyme concentrations, more
enzymes are available to catalyze the
reaction (more reactions at once)
n
tion (at
• There i
reactio
consta
s a linear relationship betwee
n rate and enzyme concentra
nt substrate concentration)

11. Substrate Concentration and Reaction Rate
• The rate of reaction increases as substrate
concentration increases (at constant enzyme
concentration)
• Maximum activity occurs when the enzyme
is saturated (when all enzymes are binding substrate)
• The relationship between reaction rate and substrate
conc. Is exponential and asymptote when the enz is
saturated.

12. Substrate Concentration and Reaction Rate
Rate• Tses as substrate concentration increases
(at constant enzyme concentration)
• Maximum activity occurs when the
enzyme is saturated (when all enzymes are
binding substrate)
• The relationship between reaction rate and
ial,
e
subst
and a
enzym
rate concentration is exponent
symptotes (levels off) when th
e is saturated

13. Products of Reaction and Reaction Rate
If products are removed as fast as they are
formed,the reaction will be 100% complete,but if
they are not removed,the reaction will remain
incomplete.

14. ACTIVATIO
N

15. ACTIVATION
 Activation is defined as the conversion of an inactive form of
an enzyme to active form which processes the metabolic
activity.
TYPES OF ACTIVATION
 Activation by co-factors.
 Conversion of an enzyme precursor.

16. ACTIVATION BY CO FACTORS
 Many enzymes are activated by co-factors.
Examples:
 DNA polymerase is a holoenzyme that catalyzes the
polymerization of de -oxyribonucleotide into a DNA strand. It
uses Mg- ion for catalytic activity.
 NADH Dehydrogenase uses NADH for its activation
Carbonic anhydrase uses Zn- ion for it‟s activation.

17. CONVERSION OF AN ENZYME
PRECURSOR
 Specific proteolysis is a common method of activating
enzymes and other proteins in biological system.
Example:
 The generation of trypsin from trypsinogen leads to the
activation of other zymogens.

18. ZYMOGEN ACTIVATION BY
PROTEOLYTIC CLEAVAGE

19. INHIBITIO
N

20. INHIBITION
o The prevention of an enzyme process as a result of interaction of
inhibitors with the enzyme.
 INHIBITORS:
Any substance that can diminish the velocity of an
enzyme catalyzed reaction is called an inhibitor.

21. TYPES OF INHIBITION
Inhibition
Reversible
Competitive Non-
competitive
Irreversible

22. REVERSIBLE INHIBITION
o It is an inhibition of enzyme activity in which the inhibiting
molecular entity can associate and dissociate from the
enzyme’s binding site.
TYPES OF REVERSIBLE INHIBITION
o There are two types:
 Competitive inhibition
 Non-competitive inhibition.

23. COMPETITIVE INHIBITION
 In this type of inhibition, the inhibitors compete with the
substrate for the active site. Formation of E.S complex is
reduced while a new E.I (Enz-inhibitor) complex is formed.

24. EXAMPLES OF COMPETITIVE INHIBITION
 Statin Drug As Example Of Competitive Inhibition:
 Statin drugs such as lipitor compete with HMG-CoA(substrate)
and inhibit the active site of HMG CoA-REDUCTASE (that
bring about the catalysis of cholesterol synthesis).

25. • Malonate is a competitive inhibitor of
succinate dehydrogenase
- it has a structure that is similar to
succinate
-inhibition can be reversed by
adding succinate.

27. Competitive Inhibitors-Effect on reaction velocity versus
substrate conc. plot and lineweaver-burk plot

28. Competitive Inhibitors Used As Drugs-Antimetabolites
Competitive Inhibition (substrate analogue inhibition),can be reversed
by adding an excess of normal substrate.
It is used as chemotherapy against bacterial and malignant cells and
viruses.
Some naturally occuring substances and their antimetabolites are:
1-For paraaminobenzoic acid(PABA)-sulfonamides(septran)-inhibit
formation of folic acid by bacterial cells-kill them-cure infectuon.
2-For xanthine-Allopurinol -inhibit formation of uric acid-cure
hyperuricemia.
3-Uracil-5-Fluorouracil -inhibit RNA synthesis-kill RNA viruses.
4-Pteroylglutamic acid-Aminopterin -inhibit DNA synthesis-used as
cytotoxic agents to cure malignant conditions.

29. Non-COMPETITIVE INHIBITION
 In this type of inhibition, inhibitor does not compete with the
substrate for the active site of enzyme instead it binds to
another site known as allosteric site.

30. Reversible Inhibitors (Noncompetitive Inhibition)
• A noncompetitive Non inhibitor has a
structure that is different than that of the
substrate
-it binds to an allosteric site
rather than to the active site
-it distorts the shape of the enzyme, which
alters the shape of the active site and
prevents the binding.
-It can bind to ES-complex as well.

33. Non-COMPETITIVE INHIBITION
EXAMPLE:
EDTA(Ethylene diamin tetra acetic acid):used in labs as anticoagulant
in blood samples,it bind all the Ca++ in blood sample,therefore inhibit
all enzymes which need Ca++ as co-factor for their activation.
FLOURIDE IONS: Remove Mg++ needed for many enzymes
activation e.g. Enolase in glycolysis.
Replacement of these ions(Ca++ & Mg++) can result in reappearance
of the activity of the enzymes.

34. Non-Competitive inhibition-Effect on reaction velocity
versus substrate conc. plot and lineweaver-burk plot

35. IRREVERSIBLE INHIBITION
 This type of inhibition also called enzyme poisoning involves the
strong covalent attachment of the inhibitor to the enzyme.
 The catalytic activity of enzyme is completely lost.
 It can only be restored only by synthesizing molecules.

36. Irreversible Inhibitors
• An irreversible inhibitor destroys enzyme
activity, usually by covalent bonding with side-chain
groups in active site.

37. DFP=DichloroFlouroPhosphate

38. EXAMPLES OF IRREVERSIBLE
INHIBITION
 Lead: inhibit Ferrochelatase,enzyme irreversibly in heme
synthesis.
 SUICIDE INHIBITION :
 It is an unusual type of irreversible inhibition where the
enzyme converts the inhibitor into a reactive form in its active
site.

39. Enzyme Inhibitors used as Drugs
1>Captopril -ACE inhibitor -decrease angiotensionII-used in
hypertension
2>Acetazolamide -inhibit carbonic anhydrase-decrease formation of
aq.humor-used in Glaucoma
3>Aspirin-NSAIDs-inhibit COX-1/2-inhibit inflamation-used in
inflamatory diseases.
4>Alpha-1-Antitripsin-inhibit Elastase-used in Emphysema
5>Omeprazole-PPI-proton pump inhibitor-inhibit HCl formation-used
in peptic ulcer disease.
6>Cardiac Glycosides-inhibit Na-K ATPase in heart-increase force of
myocardial contraction-used in heart failure

40. ENZYME REGULATION

41. ENZYME REGULATION
Regulation of reaction velocity of enzyme is essential to coordinate
numerous metabolic processes.
By 3-ways:
1-ALLOSTERIC ENZYMES
2-BY COVALENT MODIFICATION
3-ENZYME INDUCTION AND REPRESSION

42. REGULATION OF ENZYMES -Allosteric Enzyme
Allosteric Enzymes:
These enzymes have Alosteric sites,where Effector molecules
bind,noncovalently,which alter the affinity of enzyme for its substrate.
Negative effectors: Decrease affinity-Inhibit enzyme activity.
Positive effectors: Increase affinity-stimulate enzyme activity.
Important allosteric effectors produced in response to certain
hormones are: c AMP,c GMP,DAG,IP3,Ca++ and Calmodulin.
Two types:
Homotropic effectors:
Heterotropic effectors:

43. Homotropic effectors
Homotropic effectors:
When the substrate itself serves as an effector,
Mostly substrate act as positive effector.
Example,
In Glycolysis,more glucose activate more hexokinase
Glucose+ATP--------------------G-6-P +ADP
Hexokinase
Tripsin once activated by Enterokinase,itself can activate other
tripsinogen molecules.

44. Heterotropic Effectors
The effector may be different from the substrate.e.g. feed back
inhibition by the end product,which is different from substrate.
Glucose+ATP--------------------G-6-P +ADP
Hexokinase

46. Positive and negative effectors can affect either Vmax or Km or both.
A-Vmax is altered
B-The sub. Conc.that give ½ Vmax (K0.5) is altered.

47. REGULATION OF ENZYMES-By Covalent Modification
It is by addition or removal of phosphate groups (by protein kinases+ATP
/phosphatases) from specific serine,threonine or tyrosine residues of the
enzyme.
Response of enzyme to phosphorylation:
Phosphorylated form may be more or less active than unphosphorylated
enzyme.
e.g.phosphorylation of glycogen phosphorylase increase activity,
while addition of phosphate to glycogen synthase decrease activity.

49. REGULATION BY-
Induction and repression of enzyme synthesis
The regulatory mechanisms described above modify the activity of
existing enzyme molecules.
Cells can also regulate the amount of enzyme present by altering the
rate of enzyme degradation or, more typically, the rate of enzyme
synthesis.
The increase (induction) or decrease (repression) of enzyme synthesis
leads to an alteration in the total population of active sites, under
selected physiologic conditions.
For example, elevated levels of insulin as a result of high blood
glucose levels after meal cause an increase in the synthesis of key
enzymes involved in glucose metabolism.

50. Induction n Repression of Enzymes
In some cases drugs are used to activate or
induce a certain enzyme action.
Example is phenobarbitone used to decrease
raised unconjugated billirubin in whome UDP-
glucuronyl transferase is defficient (newborn
babies) in hepatocytes.

51. MECHANISMS FOR REGULATING
ENZYME ACTIVITY

52. PROPERTIES OF ENZYMES

53. 1-Specificity
2-Protein nature
3-The direction of enzyme reaction
4-Proenzyme/Zymogens
5-Induction/Repression of enzymes
6-Isoenzymes/Isozymes
7-Location of enzymes

54. ENZYME
SPECIFICITY

55. ENZYME SPECIFICITY
 Enzymes are highly specific in nature, interacting with one or
few substrates and catalyzing only one type of chemical
reaction.
 Substrate specificity is due to complete fitting of active site
and substrate .
Example:
 Oxydoreductase do not catalyze hydrolase reactions and
hydrolase do not catalyze reaction involving oxidation and
reduction.

56. • Enzymes have varying degrees of specificity for
substrates
• Enzymes may recognize and catalyze:
- a single substrate
- a group of similar substrates
- a particular type of
bond

57. TYPES OF ENZYME SPECIFICITY
 Enzymes show different degrees of specificity:
 Absolute specificity.
 Group specificity.
 Bond specificity.
 Optical or stereo-specificity.
 Dual specificity.

58. GROUP SPECIFICITY
 In this type of specificity, the enzyme is specific not only to the
type of bond but also to the structure surrounding it.
Example:
 Pepsin is an endopeptidase enzyme, that hydrolyzes central
peptide bonds in which the amino group belongs to aromatic
amino acids e. g phenyl alanine, tyrosine and tryptophan.

59. BOND SPECIFICITY
 In this type, enzyme acts on substrates that are similar in
structure and contain the same type of bond.
Example :
 Amylase which acts on α-1-4 glycosidic ,bond in starch
dextrin and glycogen, shows bond specificity.

60. ABSOLUTE / SUBSTRATE SPECIFICITY
 In this type of specificity ,the enzymes acts only on one
substrate
Example :
 Uricase ,which acts only on uric acid, shows substrate
specificity.
 Maltase , which acts only on maltose, shows substrate
specificity.

61. OPTICAL / STEREO-SPECIFICITY
 In this type of specificity , the enzyme is not specific to
substrate but also to its optical configuration
Example:
 D amino acid oxidase acts only on D amino acids.
 L amino acid oxidase acts only on L aminoacids.

62. DUAL SPECIFICITY
 There are two types of dual specificity.
may act on one substrate by two different The enzyme
reaction types.
Example:
 Isocitrate dehydrogenase enzyme acts on isocitrate (one
substrate) by oxidation followed by decarboxylation(two
different reaction types) .

63.  The enzyme may act on two substrates by one reaction type
Example:
• Xanthine oxidase enzyme acts on xanthine and
hypoxanthine(two substrates) by oxidation (one reaction type)
DUAL SPECIFICITY

64. Isoenzymes
• Isoenzymes are different forms of an enzyme that
catalyze the same reaction in different tissues in body.
• They have slight variations in the amino acid sequences
of the subunits of their quaternary structure.
• Different organs commonly contain characteristic proportions
of different isoenzymes.
• The pattern of isoenzymes found in the plasma serve as a
means of identifying the site of tissue damage.
• For example, lactate dehydrogenase (LDH), which
converts lactate to pyruvate, consists of five
Isoforms.

65. Isoenzymes• I f enzyme that catalyze the same reaction in
different tissues in the body
- they have slight variations in the amino acid
sequences of the subunits of their
quaternary structure
• For example, lactate dehydrogenase (LDH), which
converts lactate to pyruvate, consists of five
iso
enzyme
s

66. ISOENZYMES AND CARDIAC DISEASES
plasma levels of creatine kinase (CK) are commonly determined in the
diagnosis of myocardial infarction (MI). CK occurs as three
isoenzymes.
Each isoenzyme is a dimer composed of two polypeptides
(called B and M subunits) associated in one of three combinations:
CK1= BB- in brain
CK2 = MB In cardiac muscle -
CK3 = MM- in skeletal muscle
Each CK isoenzyme shows a characteristic electrophoretic mobility :

68. INTRACELLULAR AND
EXTRACELLULAR ENZYMES
o Intracellular enzymes are synthesized and retained in the cell
for the use of cell itself.
o They are found in the cytoplasm, nucleus, mitochondria and
chloroplast.
Example :
 Oxydoreductase catalyses biological oxidation.
 Enzymes involved in reduction in the mitochondria.
in the cell buto Extracellular enzymes are synthesized
secreted from the cell to work externally.
Example :
 Digestive enzyme produced by the pancreas, are not used by
the cells in the pancreas but are transported to the
duodenum.

69. INTRACELLULAR ENZYMES

71. DIAGNOSTIC VALUE OF SERUM ENZYME LEVELS
Creatine Kinase: increase in myocardial infarction
Lactate Dehydrogenase: increase in acute myocardial infarction and
acute liver demage
Alkaline phosphatase: increase in bone n liver
diseases,rickets,obstrutive jaundice etc.
Acid phosphatase: increase in prostatic carcinoma
AST: increase in myocardial infarction n liver demage
ALT: increase in acute liver demage
Amylase/lipase: increase in pancreatitis n parotitis
Γ Glutamyl transpeptidase(GTT): increase in hepatobilliary obstruction
And in alcohol induced liver demage

72. Diagnostic
Enzymes
• The levels of diagnostic enzymes in the
blood can be used to determine the
amount of damage in specific tissues

73. DIAGNOSIS OF MYOCARDIAL INFARCTION
BY CK2(MB) ISOENZYME:
It is specific for MI,appears in blood 4- 8 hours,following onset of chest
pain,reaches a peak at 24 hrs,returns to baseline after 2-3 days.
TRPONINE T n I are newest markers for MI:
cTnI is highly sensitive and spefific for cardiac muscle demage.It
appears in plasma within 4-6 hrs,peaks in8-28hrs,and remain elevated
for 3-10 days.
LDH:peak on
AST:

75. THERAPEUTIC USES OF ENZYME (as drugs)
Enzymes for thrombolysis: Streptokinase,convert plasminogen to
plasmin-lysis of fibrin clot and Urokinase,activate firinolysine system
Enzymes for cancer treatment: L.Asparginase, to treat lymphoblastic
leukemia in children
Enzyme substitution in digestive disturbances: contain
proteases,lipases and amylases
Blood coagulation:Thrombin is locally used to stop bleeding
Hyaluronidase: used locally to decrease swelling,in sprains,hematoma
n thrombophlebitis
Trypsin n chymotrypsin: locally/orally used to degrade necrotic
tissue,pus for wound clearing and healing

76. ASSIGNMENT FOT TODAY
1-DIFFERENTIATE B/W:
Ribozymes and zymogens
and isoenzymes
2-Enlist:
diagnostic and therapeutic
uses of enzymes