Enzymes, terminology, factors affected activity, classification, kinetic, enzymes uses

1. 1

2. Enzymes
Dr
Mohamed Mostafa Omran
Faculty of Sciences, Helwan University
2

3. Introduction to Biochemistry

4. Biochemistry
• Chemistry of living organisms.
• Each second/ 8,000 trillion reactions in
our bodies

5. Biomolecules
•
•
•
•
•
•
Carbohydrates
Lipids
Proteins
Nucleic acid
Vitamins
Enzymes

6. Enzymes
Made of protein
Present in
all living cells
Enzymes
Biological
catalysts
Converts substrates
into products
Affected by cellular
conditions: any condition
that affects protein
structure temperature, pH,
salinity
Increase the rate of
Remain unchanged
chemical reactions
by chemical reaction
proceeding from10^3–10^8 times
faster than uncatalyzed reactions.

7. Properties of enzymes
• Reaction specific
– each enzyme works with a specific substrate
• chemical fit between active site & substrate
– H bonds & ionic bonds
• Not consumed in reaction
– single enzyme molecule can catalyze thousands or
more reactions per second
• enzymes unaffected by the reaction
• Affected by cellular conditions
– any condition that affects protein structure
• temperature, pH, salinity

8. Specificity of the Enzyme
• For enzyme and substrate to react, surfaces of each must
be complementary
• Enzyme specificity: the ability of an enzyme to bind only
one, or a very few, substrates thereby catalyzing only a
single reaction
• Compare these 2 reactions:
• Urease is VERY
Specific or has a
HIGH DEGREE of
Specificity

9. Specificity of Enzymes One of the properties of enzymes that makes them so important
as diagnostic and research tools is the specificity they exhibit relative to the reactions
they catalyze.
In general, there are four distinct types of specificity:
1. Absolute specificity - the enzyme will catalyze only one reaction.
2. Group specificity - the enzyme will act only on molecules that have specific
functional groups, such as amino, phosphate and methyl groups.
3. Linkage specificity - the enzyme will act on a particular type of chemical bond
regardless of the rest of the molecular structure.
4. Stereochemical specificity - the enzyme will act on a particular steric or optical
isomer. Though enzymes exhibit great degrees of specificity, cofactors may serve many
apoenzymes.

10. Enzyme Specificity

11. Function of Enzymes
•
Enzymes speed up the rate of chemical
reactions in the body; both breaking
down (e.g.: starch into maltose)
and building up reactions. (e.g: amino
acids into proteins).
•
Enzymes lower the activation energy
required to start a chemical reaction

12. Characteristics of Enzymes
• Enzymes are highly specific
in action.
• Enzymes remain chemically
unchanged at the end of
the reaction.
• Enzymes are required in
minute amounts.

13. is a non-protein part firmly attached to the apoprotein
13

14. Cofactors are bound to the enzyme
for it to maintain the correct
configuration of the active site
14

15. How Coenzymes work
• A coenzyme is
required by some
enzymes
– An organic molecule
bound to the enzyme
by weak interactions /
Hydrogen bonds
– Most coenzymes carry
electrons or small
groups
– Many have modified
vitamins in their
structure

16. Water-Soluble Vitamins and Their Coenzymes

17. 17

18. Turnover number (Enzyme Unit)
The “turnover number” is the number of
substrate molecules converted into product
by an enzyme molecule in a unit time under
optimal condition of measurement.
18

19. Zymogens (proenzymes)
• Digestive and coagulation enzymes are secreted in inactive
forms, zymogens or proenzymes.
• Zymogens are activated by trimming of a short peptide
blocking the active site, or by covalent modification of the
zymogen.
19

20. Mechanism of activation
* The mechanism of activation may involve unmasking of a
polypeptide chain that may be blocking or masking the active
centers of apoenzyme.
* Proteolytic enzymes are secreted inactive to prevent
digestion of protein of the cell that synthesized them.
Activation takes place by:
HCL
1- pH – changes: Pepsinogen
Pepsin
trypsin
2- Auto-activation: Trypsinogen
Trypsin
Entrokinase
3- By other enzymes: Trypsinogen
Trypsin

21. 21

22. ase
Protein
Substrate Name + -ase

23. Carbohydrate
ase

24. Terminology of enzymes
1-Some enzymes still retain their old names as digestion enzymes still use –in
pepsin, trypsin.
2-End in –ase:
Identifies a reacting substance
lipase - reacts lipid
3-Describes function of enzyme:
oxidase – catalyzes oxidation
hydrolase – catalyzes hydrolysis
24

25. Terminology of enzymes
Nomenclature systems are
4. The trivial name: which give no indication of the function of the
enzyme, are commonly used.
In some cases, the trivial name is composed of the name of the substrate
involved, the type of the reaction catalyzed, and the ending – ase.
Lactate + dehydrogenation + ase = lactate dehydrogenase
5. The systematic name:
It is made up of the names of substrates acted upon , coenzyme involved in
the reaction , the type of reaction catalyzed+ ase
eg Lactate NAD+ Oxidoreductase (the old name was lactate
dehydrogenase).
25

26. Classification of enzymes: six classes according to reaction type
(Each class comprises other subclasses)
Enzyme class
General scheme of reaction
1. Oxidoreductases
Ared + Box  Aox + Bred
2. Transferases
A-B + C  A + C-B
3. Hydrolases
A-B + H2O  A-H + B-OH
4. Lyases
A-B  A + B
5. Isomerases
A-B-C  A-C-B
6. Ligases (synthetases)
A + B + ATP  A-B + ADP + Pi
(reverse reaction: synthases)
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27. 1 Oxidoreductases
catalyze the oxidation or reduction of substrate
subclasses:
1. dehydrogenases catalyze transfers of two hydrogen atoms
2. oxygenases catalyze the incorporation of one/two O atoms
into the substrate (monooxygenases, dioxygenases)
3. oxidases catalyze transfers of electrons between substrates (e.g.
cytochrome c oxidase, ferroxidase)
4. peroxidases catalyze the breakdown of peroxides
Example: lactate + NAD+  pyruvate + NADH + H+
lactate dehydrogenase
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28. 2 Transferases
catalyze the transfer of a group from one to another substrate
subclasses:
• aminotransferases, methyltransferases,
• phosphomutases – the transfer of the group PO32– within molecule
• kinases phosphorylate substrate by the transfer of phosphoryl group
PO32– from ATP (e.g. hexokinases, proteinkinases)
Example: glucose + ATP

glucose 6-P + ADP
glucokinase
28

29. 3 Hydrolases
catalyze the hydrolytic splitting of esters, glycosides, amides, peptides etc.
subclasses:
• esterases (lipases, phospholipases, ribonucleases, phosphatases)
• glycosidases (e.g. sucrase, maltase, lactase, amylase)
• proteinases and peptidases (pepsin, trypsin, cathepsins,
dipeptidases, carboxypeptidases, aminopeptidases)
• amidases (glutaminase, asparaginase)
• ATPases (split anhydride bonds of ATP)
Example: glucose 6-P + H2O  glucose + Pi
glucose 6-phosphatase
29

30. 4 Lyases
catalyze non-hydrolytic splitting or forming bonds C–C, C–O, C–N, C–S through
removing or adding, respectively, a small molecule (H2O, CO2, NH3)
Some frequent recommended names:
• ammonia lyases (e.g. histidine ammonia lyase: histidine  urocanate + NH3)
• decarboxylases (amino acid  amine + CO2)
• aldolases (catalyze aldol cleavage and formation)
• (de)hydratases (carbonate dehydratase: CO2 + H2O  H2CO3)
Example: fumarate + H2O 
L-malate
fumarate hydratase
Fumarate is hydrated to malate in a freely reversible reaction
catalyzed by fumarase (also called fumarate hydratase)
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31. 31

32. 6 Ligases
catalyze formation of high-energy bonds C–C, C–O, C–N
in the reactions coupled with hydrolysis of ATP
Frequent recommended names:
carboxylases
synthetases
(e.g. glutamine synthetase: glutamate + ATP + NH3  glutamine + ADP + Pi)
Example: pyruvate + CO2 + ATP + H2O  oxaloacetate + ADP + Pi
pyruvate carboxylase
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33. !
Three enzymes have something to do with phosphate
Enzyme (Class) Reaction scheme / Reaction type
Kinase
substrate-OH + ATP  substrate-O-P + ADP
(Transferase)
phosphorylation = transfer of phosphoryl PO32– from ATP to substrate
Phosphatase
(Hydrolase)
substrate-O-P + H2O  substrate-OH + Pi
the hydrolysis of phosphoester bond
(glycogen)n + Pi  (glycogen)n-1 + glucose 1-P
Phosphorylase inosine + Pi  hypoxanthine + ribose 1-P
(Transferase)
phosphorolysis = the splitting of glycoside bond by
phosphate = transfer of glucosyl to inorganic phosphate
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34. !
Distinguish:
Three types of lysis (decomposition of substrate)
the decomposition of substrate by water, frequent in intestine:
Hydrolysis
sucrose + H2O  glucose + fructose
(starch)n + H2O  maltose + (starch)n-2
Phosphorolysis
the cleavage of O/N-glycoside bond by phosphate:
(glycogen)n + Pi  (glycogen)n-1 + glucose 1-P
the cleavage of C-C bond by sulfur atom of coenzyme A
Thiolysis
in β-oxidation of FA or ketone bodies catabolism
RCH2COCH2CO-SCoA + CoA-SH  RCH2CO-SCoA + CH3CO-SCoA
34

35. 0
Time
Salt concentration
35

36. reaction rate
Temperature
37°
temperature

37. •
Factors affecting enzyme
Temperature
function
– Optimum T°
• greatest number of molecular collisions
• human enzymes = 35°- 40°C
– body temp = 37°C
– Heat: increase beyond optimum T°
• increased energy level of molecules disrupts bonds in
enzyme & between enzyme & substrate
– H, ionic = weak bonds
• denaturation = lose 3D shape (3° structure)
– Cold: decrease T°
• molecules move slower
• decrease collisions between enzyme & substrate

38. Enzymes and temperature
• Different enzymes function in different
organisms in different environments
reaction rate
human enzyme
hot spring
bacteria enzyme
37°C
temperature
70°C
(158°F)

39. pH
trypsin
reaction rate
pepsin
pepsin
trypsin
0
1
2
3
4
5
6
pH
7
8
9
10
11
12
13
14

40. Factors affecting enzyme function
• pH
– changes in pH
• adds or remove H+
• disrupts bonds, disrupts 3D shape
– disrupts attractions between charged amino acids
– affect 2° & 3° structure
– denatures protein
– optimal pH?
• most human enzymes = pH 6-8
– depends on localized conditions
– pepsin (stomach) = pH 2-3
– trypsin (small intestines) = pH 8
0 1 2 3 4 5 6 7 8 9 10 11

41. reaction rate
Salinity
salt concentration

42. Factors affecting enzyme function
• Salt concentration
– changes in salinity
• adds or removes cations (+) & anions (–)
• disrupts bonds, disrupts 3D shape
– disrupts attractions between charged amino acids
– affect 2° & 3° structure
– denatures protein
– enzymes intolerant of extreme salinity
• Dead Sea is called dead for a reason!

43. 43

44. 44

45. Substrate Concentration
As substrate concentration
increases, the rate of reaction
increases (at constant enzyme
concentration).
• the enzyme eventually
becomes saturated giving
maximum
activity.
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47. 47

48. 48

49. 49

50. Important conclusion about Km
1.
2.
3.
4.
Substrate are usually in physiological fluids in amounts nearly equal to Km values.
Km is a constant characteristic of an enzyme and its substrate. Km reflect the affinity
of the enzyme for the substrate.
Low Km reflect high affinity of the enzyme for substrate
High Km reflect low affinity of the enzyme for substrate
•
Glucose
•
•
Hexokinase is more active than glucokinase
because the amount of glucose (substrate) needed to produce ½ V max in case of
hexokinaes is less than in case of glucokinase
i.e Km of hexokinase is less than glucokinase.
•
Hexokinase or glucokinase
Glucose 6 phosphatase
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51. 51

52. 52

53. 53

54. Uncompetitive

55. 55

56. 56

57. Classes of Inhibition
Two real, one hypothetical
• Competitive inhibition - inhibitor (I) binds only to E, not to ES
• Noncompetitive inhibition - inhibitor (I) binds either to E and/or
to ES
• Uncompetitive inhibition - inhibitor (I) binds only to ES, not to
E. This is a hypothetical case that has never been documented
for a real enzyme, but which makes a useful contrast to
competitive inhibition

58. 58

59. 59

60. 60

61. 61

62. 62

63. Mechanisms for Regulating Enzyme Activity
1. Allosteric Enzymes
• Allosteric means "other site" or "other structure".
• Enzymes whose activity can be changed by molecules (effector
molecules) other than substrate.
• The interaction of an inhibitor at an allosteric site changes the
structure of the enzyme so that the active site is also changed.

64. 2 Processes Involving the Allosteric Enzyme
1. Negative Allosterism: effector binding sites alters
the shape of the active site of the enzyme making
it to an inactive configuration.

65. 2. Positive Allosterism
- effector binding sites that alters the shape
of inactive site of enzyme to an active
configuration.
Therefore, binding of the effector molecule
regulates enzyme activity by determining
whether it will be active or not.

66. Vo vs [S] for Allosteric Enzymes

67. 2. Feedback Inhibition
• An enzyme regulation process in which formation of a
product inhibits an earlier reaction in the sequence. It
controls the allosteric enzymes.

68. 3. Proenzymes (Zymogen)
• The inactive form of enzyme which can be activated
by removing a small part on their polypeptide chain.
• Mostly are the digestive enzymes and blood clotting
enzymes.
• Why is it that digestive enzymes are in inactive state
before it becomes active?
• This is necessary to prevent digestion of pancreatic
and gastric tissues.

69. Zymogen
• Pepsinogen
• Trypsinogen
• Prothrombin
Active Form of Enzyme
• Pepsin
• Trypsin
• Thrombin

70. 4. Phosphorylation:
• Some enzymes may be regulated by addition or
removal of phosphate groups from enzymes
• phosphate is transferred from an activated donor
(usually ATP) to an amino acid on the regulatory
enzyme, is the most common example of this type of
regulation.

71. Phosphorylation
• Phosphorylation reactions are catalyzed by protein kinase
and removed by protein phosphatase.
• The phosphorylated enzyme may be more or less active the
unphosphorylated enzymes.
• Phosphorylation of glycogen phosphorylase enzyme result
in increase enzyme activity.
• Phosphorylation of glycogen synthase enzyme result in
less enzyme activity.
71

72. 5. Isoenzymes: are enzymes catalyze the same chemical reaction
(identical functions), isolated from different tissues, have same
number but different amino acid sequence.
Differences may be:
– A.acid sequence
– Some covalent modification
– 3-D structure
– The existence of isozymes permits the fine-tuning of
metabolism to meet the particular needs of a given tissue or
developmental stage.
– Isoenzymes e.g
– Creatine kinase (CK)
– Creatine phosphokinase CPK
– alkaline phosphatase (ALP)

73. Percentage of Enzymes Usage in Industries:

74. Detergentes
• Contain:
- lipase: greasy stains
- protease: eggs, blood
• Advantage: they work at lower
temperatures, so less water heating is
needed, and clothes don´t shrink.

75. Food industry
• Fruit juices: are extracted using pectinase. It
breaks down pectin and is much easier to squeeze
juice from the fruit. It also makes the juice clear
rather than cloudy.
• Biscuits: - isomerase: converts glucose to
fructose, which is sweeter so less needs to be used
in slimmers biscuits.
- protease: softens glutens, making the
roller of biscuits easier

76. Three utilizations of enzymes in medicine
1. enzymes as indicators of pathological
condition
2. enzymes as analytic reagents in clinical
chemistry
3. enzymes as drugs
76

77. Enzymes as tumor markers
Enzyme
Disease
Serum acid phosphatase
Cancer prostate
Serum Alkaline phosphatase
*Metastasis in liver, jaundice due to
carcinoma head of pancreas, osteoblastic
metastasis in bones
*Metastasis, or metastatic disease, is the
spread of a cancer from one organ or part to
another non-adjacent organ or par
Serum LDH
Advanced malignancies and Leukemias
Β- Glucuronidase
Cancer of urinary bladder
Leucine Amino Peptidase (LAP)
Liver cell carcinoma
Neuron specific Enolase
Malignancies of nervous tissue and brain
77

78. Summary-Enzymes as diagnostic markers
NAME OF THE ENZYME
Conditions in which level of activity
in serum is elevated
Aspartate Amino transferase (AST)
Serum glutamate-oxaloacetate
transaminase (SGOT)
Myocardial infarction, Liver disease
especially with liver cell damage
Alanine Amino transferase (ALT)
Serum glutamate-pyruvate
transaminase (SGPT)
Liver disease especially with liver cell
damage
Alkaline Phosphatase (ALP)
Liver disease- biliary obstruction
Osteoblastic bone disease-rickets
Acid Phosphatase (ACP)
Prostatic carcinoma
 glutamyl Transferase ( GT)
Liver disorder like liver cirrhosis and
alcoholism
Creatine kinase (CK)
Myocardial infarction and skeletal
muscle disease(muscular dystrophy
Lactate Dehydrogenase (LDH)
Myocardial infarction, other diseases
like liver diseases, some blood
diseases
 Amylase
Acute pancreatitis
78

79. Enzymes as diagnostic reagents
Enzyme
Used for testing
Urease
Urea
Uricase
Uric acid
Glucose oxidase
Glucose
Cholesterol oxidase
Cholesterol
Lipase
Triglycerides
Alkaline phosphatase
ELISA
Horse radish Peroxidase
ELISA
Restriction endonuclease
Recombinant DNA technology
Reverse transcriptase
Polymerase chain reaction
79

80. Enzymes as therapeutic agents
Enzyme
Therapeutic Application
Trypsin, lipase and amylase
Pancreatic insufficiency
Asparaginase/Glutaminase
Acute lymphoblastic leukemias
Hyaluronidase
Enhanced local anesthesia and for easy
diffusion of fluids
Papain
Anti inflammatory
Serratopeptidase
Pain killer and Anti inflammatory
Chymotrypsin
Pain killer and Anti inflammatory
Alpha- 1 Antitrypsin
Deficiency and Emphysema
80