Basics and applied importance of Enzymes in Medical Science, especially for nursing faculty

1. ENZYMOLOGY
Prof (Dr) Viyatprajna Acharya
MD, PhD
KIMS, KIIT (DU)

2. What shall we learn?
Why should we learn about enzymes?
Why nursing staff should know about them?
Who thought about them first?- History
How do we define them?
How do we name them?- The classification

4. Definition
Enzymes are “Biocatalysts”, synthesized by living
cells and highly specific in their action.
They are:
Mostly proteins (Exception- Ribozymes)
Heat labile
Soluble in water
Colloidal
Precipitated by precipitation reaction
Contain 16% weight as nitrogen

5. Classification & Nomenclature
A. Recommended name
B. Systematic name
IUBMB
Unambiguous & Informative
But Cumbersome
6 major classes
Class.Subclass.sub-subclass.substrate

7. OTHLIL
Oxidoreductases→ Transfer of H, O or e-
Transferases → Transfer of gr other than H
Hydrolases → Cleave bond & add H2O
Lyases → Cleave bond without adding H2O
Isomerases → Intramolecular transfers
Ligases → ATP dependent condensation of 2 molecules

8. 1. Oxidoreductases
AH2 + B →A + BH2
Alcohol+ NAD+ ADH Aldehyde + NADH +H+
• Dehydrogenases (hydride transfer)- ADH
• Oxidases (electron transfer to molecular oxygen)- L-
and D- AA oxidase
• Oxygenases (oxygen transfer from molecular
oxygen)- oxygenase, mono- & di-oxygenases,
Cytochrome oxidase
• Peroxidases (electron transfer to peroxide)-
Glutathione peroxidase

9. 2. Transferases
A-R + B → A + B-R
• transfer of an atom or group of atoms
(e.g. acyl-, alkyl- and glycosyl- ), between
two molecules, but excluding such
transfers as are classified in the other
groups (e.g. Oxidoreductases and
Hydrolases).
• Ex- Aminotransferases, all kinases,
transmethylases
• Hexose + ATP Hexokinase Hexose-6-P +
ADP

10. 3. Hydrolases
Cleavage of ester, ether, peptide or
glycosidic bond by addition of H2O
Ach + H2O acetylcholine esterase Choline +
Acetate
Ex: All digestive enzymes, lipase, pepsin,
Trypsin, ALP, Urease

11. 4. Lyases
Cleave bond without addition of H2O
Fructose-1,6-BP Aldolase Glyceraldehyde-3-
P
+ Dihydroxyacetone P
Ex: Fumarase, Histidase, HMG CoA lyase

12. 5. Isomerases
Can produce optical, geometrical or
positional isomers of substrates
Gly-3-P Triose P isomerase DHAP
Ex: Racemase, Epimerase

13. 6. Ligases (synthetases)
ATP dependent condensation of two
molecules
Acetyl CoA + CO2 + ATP Acetyl CoA Carboxylase
Malonyl CoA + ADP + Pi
Synthases and synthetases are different
!!!
Synthatase- need ATP; Synthase- no ATP
Ex: Glycogen synthase, ALA synthase

16. Some Terminologies
Active Site
 Region where substrate binds
 Occupies a very small portion of the enzyme
 Situated in a crevice or a cleft
 During binding specific groups realign themselves to fit
exactly
 Substrate binds by non-covalent bonds
 AA or grs. That directly participate in binding are
known as catalytic residues
 Sometimes catalytic site and substrate binding site may
be different
 Coenzymes and cofactors are a part of the catalytic site
 Serine- frequently present

17. Active site

18. Specificity
• Highly specific
• Interacting with one or few substrates
• Catalyze only one reaction

19. Types of specificity
• Absolute specificity
• Group specificity
• Bond specificity
• Stereo specificity

20. Cofactor/ Coenzyme
• Holoenzyme → Apoenzyme + Coenzyme
(Active enzyme) (Protein part) (Non-Prot.
part)
• Some enzymes require Metal (Zn, Fe),
organic molecules (coenzymes)
• Inorganic ions- Activators

21. Characteristics of coenzymes
I. When cofactor is some organic substance
II. Group is transferred from or accepted by the
coenzyme
III. Heat stable
IV. Low MW
V. Combine loosely with enzyme
VI. Separated by dialysis
VII. Reaction complete→ Coenzyme released →
Goes to other reaction site

22. Prosthetic group
• When cofactor (collectively includes coenzymes
and metal ions) is strongly bound to the
apoenzyme by covalent or non-covalent forces
• Ex: PLP, FMN, FAD, TPP, Biotin;
metal ions of Co, Cu, Mg, Mn, Se, and Zn
• Metals are most common
• 1/3rd enzymes contain metals- Metallozymes
/Metallo-enzymes
• Metals as cofactors- Metal activated enzymes

23. Metallo -enzymes
Metal Metal containing enzyme
Zn Carbonic anhydrase, Carboxypeptidase, ADH
Mg Hexokinase, PFK, Enolase, Glu-6-Phosphatase
Mn Phospho gluco mutase, Hexokinase, Enolase,
Glycosyl transferase
Cu Tyrosinase, cytochrome oxidase, SOD, Lysyl
oxidase
Fe cytochrome oxidase, catalase, peroxidase,
Xanthine oxidase
Ca Lecithinase, lipase
Se Glutathione peroxidase, Deiodinase
Mo Xanthine oxidase

24. Enzymology contd………….
He who angers you , conquers you.
Elizabeth Kenny

25. How enzymes work???
 Enzymes provide an alternate,
energetically favorable pathway different
from uncatalyzed reaction
 The active site chemically facilitates
catalysis

26. A. Energy changes occurring during reaction
A ↔ T* ↔ B
Energy barrier separates reactants and products.
Energy barrier- Free energy of activation
↓
Energy difference between reactants and high energy
intermediate T*

27. a) Free energy of activation
 Uncatalyzed reactions- high EA
 Rate of reactions thus slow

28. Visualization of the transition state

29. Koshland’s Induced fit model
Substrate induces conformational changes in enzyme

31. Factors affecting enzyme
action
Prof. Dr. Viyatprajna Acharya

32. Enzymes can be isolated and
properties can be studied in vitro
Factors affecting enzymes are:
1. Concentration of enzyme
2. Concentration of substrate
3. Concentration of product
4. Temperature
5. pH
6. Activators
7. Time
8. Light & radiation
9. Inhibitors

33. 1. Enzyme concentration
affecting enzyme activity
 When substrate is sufficient,
Rate of reaction is proportional to
Enzyme concentration
 Unit of enzyme activity- IU, Katal
(Kat), U, KAU

35. 2. Substrate concentration
affecting enzyme activity
E + S ↔ ES ↔ E + P
A
B
C
3 Phases
A. At low substrate
conc.– V α [S]
B. [S] not directly
proportional to V
C. Reaction
independent of [S]

36. Most of the enzymes follow
Michelis-Menten kinetics

37. MICHELIS-MENTEN EQUATION
Enzyme combines reversibly with
substrate to form ES.
Breakdown of ES to product is
irreversible.
E= Enzyme
S= Substrate
P= Product
Es= Enz- substrate complex
K1, K-1, K2= Rate constants

38. Michelis constant
Km = K1+ K2
K
1
After algebraic manipulation

39. Km/ Michelis constant
 It’s the substrate concentration (expressed in
moles/lit) at half-maximal velocity.
 50% of enzyme molecules are bound with
substrate molecules at that particular substrate
concentration
 Km is independent of enzyme concentration
 Expressed in moles/lit
 Km is a constant for an enzyme. It’s the
characteristic feature of a particular enzyme for a
specific substrate- Signature of the enzyme

40.  Km is the representative of measuring
the strength of the ES complex
Low Km – strong affinity between enzyme
and substrate
Ex: Glucokinase– Km= 10 mmol /lit
Hexokinase– Km= 0.05 mmol/lit
So what’s the inference??

41. 50% molecules of Hexokinase are
saturated even at a lower conc. Of
glucose.
 When [S] << Km → reaction is first-
order
 When [S] >> Km → reaction is zero-
order

42. Double reciprocal curve

43. Cooperative binding

44. 5. Effect of pH
pH change alters:
 Ionization states of the amino acid residues
present in the active site
 Ionization state of substrate
 May dissociate apoenzyme from cofactor
 Drastic change denatures the enzyme protein
Optimum pH is different for different enzymes
Mostly 6-8
Exception: Pepsin– 1-2
ALP– 9-10
Acid phosphatase – 4-5

45. pH affecting enzyme activity
A bell-shaped curve

46. 6. Effect of activators
 Inorganic metal ions increase enzyme
activity
Ex: Mg, Mn, Zn, Co, Cu etc
Cl - - Salivary amylase
Ca++ - Lipase

47. 7. Effect of Time
 Under ideal and optimal conditions,
the time required for an enzyme
reaction is less

48. 8. Effect of light and
radiation
 UV rays,β, γ, X- rays inactivate

50. Enzyme Inhibition
Prof. (Dr.) V.P. Acharya

52. Many different kinds of molecules inhibit
enzymes and act in a variety of ways
Enzyme inhibition
Competitive
Non-competitive
Reversible Irreversible
Uncompetitive
Suicide
Allosteric
Feedback

53. Competitive inhibition
• Inhibitor competes with the substrate for the active
site
• Inhibitor is substrate analogue
• Usually reversible
• ↑ [S] abolishes inhibition
• ↓ velocity of reaction
• ↑ Km
• Vmax unchanged

54. COMPETITIVE
INHIBITION

56. Non-competitive inhibition
• No competition between substrate and
inhibitor
• Different binding sites
• No structural similarities
• ↑ [S] doesn’t resolve the inhibition
• Usually irreversible
• May be reversible when inhibitor is
removed
• Km value unchanged
• Vmax reduces

58. Clinical significance
• Cyanide inhibits cytochrome oxidase
• F inhibits enolase- removes Mn & Mg
• Heavy metals react with –SH gr. Of BAL-
hence BAL is used in heavy metal
poisoning
Toxicological importance
• Most of the poisons- Irreversible NC
inhibitors- iodoacetate, heavy metal
poisons

59. Competitive Vs Non-competitive inhibition
Competitive
inhibition
Non-competitive
inhibition
Act on Active site May/may not be
Str of inhibitor Substrate
analogue
Not an analogue
Reversibility Reversible Mostly irreversible
↑ Substrate Inhibition relieved No effect
Km ↑ No change
Vmax Unchanged ↓
Significance Drug action Toxicological

61. How Isozymes help in Medicine??
 Diagnosis
 Prognosis
 Treatment
 Biochemical assays

62. LDH iso -enzymes
No. of
isozyme
Subunit
Make
up
Tissue of origin % in
human
serum
LDH-1 H4 Heart muscle 30
LDH-2 H3M1 RBC 35
LDH-3 H2M2 Brain 20
LDH-4 H1M3 Liver, Skeletal muscle 10
LDH-5 M4 Liver, Skeletal muscle 5

63. Clinical application of LDH
 Myocardial infarction-LDH-1> LDH-2;
flipped pattern see in MI (usually LDH-2 >
LDH-1)
↑LDH-1, LDH-2- peaks at 72hrs and
stays till 1 week
 Muscular dystrophies- ↑LDH-5
 Hepatocellular damage- ↑LDH-5
 Megaloblastic anemia, renal infarction-
↑LDH-1, LDH-2
 Cancers- ↑↑↑

64. Creatine kinase
 Normal serum level- 15-100 U/L for males;
10-80 U/L for females
 Dimer, 2 units M &B
 3 isoenzymes
 MM (CK3)- Skeletal Mm.
 MB (CK2)- Heart Mm.
 BB (CK1)- Brain

65. Clinical application of
CK
 Myocardial infarction
 Muscular dystrophies
 CK-MB peaks after AMI at 10-24 hrs, returns
to normal within 2-3 days

66. Aspartate transaminase
 SGOT
 PLP as coenzyme
 Normal serum level- 5-40 U/L
 ↑ in MI
 Moderate ↑ in liver diseases

67. Alanine Transaminase
 SGPT
 PLP is the coenzyme
 Normal level- 5-40 U/L
 ↑ level- acute hepatitis
 Moderate ↑- Chronic liver disease,
cirrhosis, hepatitis-C

68. Alkaline Phosphatase
 Found in almost all tissues
 Present in cell membrane (Ecto -enzyme)
 Hydrolyses aliphatic, aromatic &
heterocyclic compounds
 Optimum pH- 9- 10
 Normal level- 80-125 U/L
 Moderate ↑- infective, alcoholic
hepatitis, CA liver
 Very high ↑- Obstructive jaundice,
toxic hepatitis
 ↑ ↑ ↑- Bone diseases

69. Enzyme patterns in
diseases
1. Hepatic diseases: ALT
AST
ALP
NTP
GGT

70. 2. Cardiac markers:
a) Enzymatic markers
 LDH
 AST
 CK-MB
b) Non- enzymatic markers
 Troponin T & I
 Myglobin
 BNP
 Copeptin

71. 3. Muscle disease
 CK-MM
 AST
 Aldolase
4. Bone disease
ALP
5. Prostate cancer
 Prostate specific antigen
 Acid phosphatase

72. Therapeutic use of enzymes
ENZYME THERAPEUTIC
APPLICATION
Asparginase ALL
Streptokinase, Urokinase Lyse intravascular clot
Streptodornase DNAse; applied locally
Pancreatin Pancreatic insufficiency
Papain Anti-inflammatory
Alpha-1-antitrypsin Emphysema

73. Enzymes in diagnostic assays
 Glucose oxidase- glucose
 Urease- urea
 Taq Polymerase- PCR
 Reverse transcriptase- Recombinant DNA
technology
 Horse radish peroxidase- ELISA

74. For more Medical Biochemistry ppt please
visit www.vpacharya.com