Chapter 5: Enzymes Flashcards
1
Q
What are enzymes?
A
Organic substances that act as biocatalysts that increase the rate of chemical reactions.
2
Q
What is the nature of enzymes?
A
Most enzymes are protein in nature. Some are RNA in nature and are called ribozymes.
3
Q
How are enzymes produced or consumed in chemical reactions?
A
They are neither produced nor consumed in chemical reactions.
4
Q
Do enzymes change chemically at the end of a reaction?
A
No
5
Q
How do enzymes affect the equilibrium of a reaction?
A
It doesn’t affect the equilibrium constant (-G stays the same).
6
Q
How specific are enzymes in their actions?
A
They are highly specific in their action. They act on specific substrate or few related substrates.
7
Q
How are enzymes made?
A
They are produced by living cells, cellular catalysts, but can work in vivo and vitro.
8
Q
What is the amount of enzymes needed for a chemical reactions?
A
They are needed in very small amounts for a chemical reaction.
9
Q
Nomenclature old method:
A
- Pepsin
- Trypsin
- Chymotrypsin
- Rennin
10
Q
Nomenclature new method
A
- Hydrolase: substrate + ase: sucrase, glucosidase, urease, arginase.
- Other actions: substrate + action of enzyme: lactate dehydrogenase, pyruvate carboxylase, adenylate cyclase.
11
Q
Enzyme specificity
A
Enzymes are highly specific in their actions, interacting with 1 absolute specificity or few related substrates of relative specificity.
12
Q
What are enzymes highly specific?
A
Enzymes specificity is due to the nature and arrangement of the chemical groups at the catalytic site.
13
Q
What is the importance of enzyme specificity?
A
- Digestive enzymes are of low specificity allowing a few numbers of enzymes to digest all food.
- Metabolic enzymes are of high specificity to be well regulated.
14
Q
Chemical nature of enzymes is divided into?
A
- Simple protein enzymes: formed of only amino acids.
- Conjugated protein enzymes.
15
Q
Conjugated protein enzymes are divided into?
A
- Protein part (apoenzyme).
- Non protein part (cofactor).
16
Q
What enzymes are protein in nature?
A
All enzymes are proteins in nature except for ribozymes, which are RNA in nature.
17
Q
What enzymes are protein in nature?
A
All enzymes are proteins in nature except for ribozymes that are RNA in nature.
18
Q
What are holoenzymes?
A
Apoenzymes and cofactors together.
19
Q
What happens to apoenzymes alone?
A
Inactive
20
Q
Cofactors
A
- Coenzyme: prosthetic group or co-substrate.
- Metal ion: mettaloenzymes or metal activated enzymes.
21
Q
Coenzymes (organic)
A
Prosthetic group: tightly bound to apoenzyme by covalent or non-covalent forces.
Co-substrate: loosely bound to apoenzymes.
22
Q
Metal (inorganic)
A
Metalloenzyme: cation is tightly bound to the apoenzyme.
Metal activated enzymes: cation loosely bound to apoenzyme.
23
Q
Enzymes are large protein molecules that contain a small specific regions each such as:
A
- Active sites (catalyst sites) or substrate binding sites: complementary to the substrate.
- Allosteric site: binds with organic modifiers.
24
Q
Active sites
A
- Amino acids arranged in a specific manner that makes the enzyme specific to one substrate or few related substrates.
- The active site is rich in many groups such as:
- COOH
- SH
- OH
- NH2 - The active site is rich in certain amino acids:
- Serine and Cysteine
- Glutamate and aspartate
- Histidine
25
What are the theories that can explain the mechanism of the enzyme’s actions?
1. Induced fit theory.
2. Lowering the energy of activation.
26
Induced fit theory
When the substrate approaches the enzyme, the active site becomes similar to the substrates so the substrate fits into the enzyme forming an Enzyme-Substrate complex that gives enzyme+product.
27
Lowering the energy of activation
1. For any substrate to give product, it should be placed at a high energy called the energy of activation. Enzymes decrease the energy of activation.
2. Enzymes don’t affect the equilibrium constant, G stays the same.
3. G = Energy of products - substrate.
28
What are the factors that affect the rate of enzyme catalyzes reactions?
1. Concentration of substrates [S].
2. Concentration of Enzymes [E].
3. Concentration of cofactors [C].
4. Concentration of products [P].
5. Temperature
6. PH
29
How many factors change at a time?
Only 1 factor is changed at a time, while the remaining factors should remain constant.
30
When should the velocity be measured?
At the beginning of the reaction (initial velocity or Vi).
31
Concentration of a substrate [S]
1. Velocity of a reaction.
2. Km (Michaelis constant).
3. Michaelis-Menten equation.
4. Significance of Km.
5. Lineweaver- Burk plot (double reciprocal plot).
32
Velocity of a reaction
Number of substrate molecules converted per unit time.
1. Increase in [S] = Increase in velocity of a reaction, directly proportional.
2. When Vmax (maximal velocity) is reached, further increase in [S] won’t increase the velocity of a reaction.
33
Km (Michaelis constant)
Substrate concentration the produces half the Vmax.
1. At Vmax, all enzymes are saturated with substrates so further increase in [S] will not increase the reaction velocity.
2. Before Km: the reaction is responsive to the increase of [S] which will lead to a linear curve.
3. After Km: the reaction is less responsive to increased [S] which will lead to a non-linear curve.
34
Michaelis-Menten equation
Describes the behavior of many enzymes when [S] is changed.
Vi= Vmax [S]/ Km + [S]
35
Significance of Km
1. Index for the affinity of the enzymes to substrates ( the lower the Km, the higher the affinity).
2. Isoenzymes have different Km’s.
3. If there is an enzyme that binds to several substrates, each substrate will have a different Km.
4. If there is an inhibitor that decreases the binding of the substrate to the enzyme, it will decrease the affinity which will increase the Km.
36
Lineweaver-Burk plot (double reciprocal plot)
1. Michaelis-Menten equation gives a non linear (hyperbolic) curve at Vmax, and increase in [S] will not increase the velocity.
2. The curve is converted into a linear one by taking the reciprocal of both the [S] and V and plot in 1/[S] and 1/V (double reciprocal plot).
3. This will give a linear relationship so at any [S], we can obtain a velocity for the lower reaction.
37
Concentration of enzymes [E]
1. An increase in [E] will lead to an increase in the velocity until a certain point (Vmax).
2. Beyond Vmax, further increase in [E] will not increase the velocity.
3. [S] is the limiting factor.
38
Concentration of Cofactors [C]
1. Increase in [C] will increase velocity of the reaction until a certain point (Vmax).
2. Beyond Vmax, further increase in [C] will not increase the Vi.
3. The [E] is the limiting factor.
39
Concentration of products [P]
1. Increase in the concentration of products will lead to the decrease of the velocity of a reaction.
2. Decrease in the concentration of products will lead to increase of the velocity of a reaction.
40
Temperature
1. Optimum temperature of most enzymes is 37 degrees Celsius.
2. Below this temperature, a decrease in velocity will occur due to decrease in collision between the enzyme and substrate . For example, 0 C will lead to the stopping of a reaction.
3. At 70C the reaction stops due to denaturation.
41
What is the optimum temperature for plant enzymes?
50C
42
PH
1. Each enzyme has its optimum PH at which the reaction reaches maximum velocity.
2. Below or above this optimum Ph, the reaction will decrease.
3. The optimum Ph for most enzymes is between 5-9.
4. Optimum Ph for pepsin is 2.
5. At Ph 2 units above or below the optimum Ph will lead to stopping of reaction.
43
Optimum Ph for pepsin
2
44
Why can the Ph affect the catalytic activity of the enzyme?
1. Slight changes in Ph: alteration of the charges on the substrate no active site of enzyme.
2. Extreme changes in Ph: Denaturation which will lead to irreversible inhibition of the enzyme action.
45
Inhibitors
Any substance that can decrease the activity of an enzyme catalyzed reaction.
46
What are types of enzyme inhibitors?
1. Competitive inhibitors.
2. Allosteric inhibitors.
3. Other inhibitors.
47
Competitive inhibitors
1. The inhibitors is similar (structural analogue) to the substrate.
2. The inhibitor competes with the substrate for binding with the active site of the enzyme which decreases the rate of chemical reaction.
3. The inhibition depends on the concentration of the inhibitor and the substrate. An increase in the concentration of the substrate will remove the inhibition (reversible inhibition).
48
What is the effect of competitive inhibitors on Km and Vmax?
1. Km is increased.
2. No effect on Vmax.
49
What are example of competitive inhibitors?
1. Sulfonamide or sulfanilamide.
2. Allopurinol.
3. Dicumarol and warfarin.
4. Statins.
50
Sulfonamide or sulfanilamide
1. Bacteriostatic (used in treatment of bacterial infection).
2. Similar to PABA (Para amino benzoic acid) .
3. Competes with PABA for the enzyme that converts PAB to folic acid.
4. Leads to decrease in folic acid synthesis and decrease in bacterial multiplication.
51
Allopurinol
1. Used in treatment of gout (decrease Uris acid formation).
2. Similar to hypoxanthine.
3. Competes with hypoxanthine for the enzymes xanthine oxidase.
4. Decreases Utica acid formation.
52
Dicumarol and warfarin
1. Anticoagulant.
2. Similar to vitamin K.
3. Competes with vitamin K for the enzyme vitamin K reductase.
4. Decreases active form of vitamin K.
5. Decreases activation of blood clotting factors.
6. Decreases coagulation.
53
Statins
1. Competitive inhibitors to HMG CoA.
2. Competes with HMG CoA reductase ( key enzyme of cholesterol synthesis).
3. Similar (structural analogue) to HMG CoA, competes with HMG CoA for binding with HMG CoA reductase.
4. Decreases plasma cholesterol.
54
Allosteric inhibitors
1. Enzymes contain certain site known as Allosteric site.
2. Binding of organic modifier to the Allosteric site may produce conformational changes in the active site:
- decreasing the affinity of the enzyme to the substrate.
55
What is the effect of Allosteric inhibitors on Km and Vmax?
1. Km increases: decrease in the affinity of the enzyme.
2. Vmax decreases: decrease catalytic activity.
56
What is an example of Allosteric inhibitors?
ATP is an Allosteric inhibitor for PFK1 (phosphofructokinase 1).
57
What is a type of allosteric inhibitors?
Feedback inhibitors.
58
Feedback inhibitors
1. Type of Allosteric inhibition.
2. The end product of a series of reactions inhibits an enzyme early in the pathway.
3. Occurs at the earliest irreversible step unique to the particular pathway.
59
What are types of feedback inhibitions?
Short loop feedback and long loop feedback.
60
Short loop feedback.
The product inhibits the previous enzyme.
61
Long loop feedback inhibition
The product inhibits an enzyme early in the pathway.
62
Competitive inhibitors mechanism of action
Inhibitor is similar to substrate and binds to the same substrate binding site.
63
Allosteric inhibitors mechanism of action
Inhibitor is not similar to substrate and binds to Allosteric site.
64
Competitive inhibitors effect on Km
Increases
65
Allosteric inhibitors effect on Km
Increases
66
Competitive inhibitors effect on Vmax
Same
67
Allosteric inhibitors effect on Vmax
Decreases
68
Examples of competitive inhibitors (table)
1. Allopurinol is the inhibitor for xanthine oxidase.
2. Statins is the inhibitor for HMG CoA reductase.
69
Examples of Allosteric inhibitors (table)
1. ATP inhibitor for PFK1
70
What are types of other inhibitors?
1. Inhibitors that exert their effect on cofactors or prosthetic group.
2. Inhibitors that exert their effects on apoprotein part of the enzyme.
71
Inhibitors that exert their effects on cofactors or prosthetic group.
1. Fluoride.
2. Cyanide and carbon monoxide.
72
Fluoride
1. Inhibits enzymes that require Ca2+ and Mg2+.
2. Binds and chelates Mg+2.
- Inhibits enclave that leads to inhibition of glycolysis.
73
Cyanide and carbon monoxide
1. Binds to Fe+2.
2. Inhibits cytochrome oxidase.
74
Inhibitors that exert their effects on apoprotein part of the enzyme
1. Nonspecific inhibitors.
2. Either anti enzymes, factors that denature protein or that block chemical groups in the active site (enzyme poison).
75
Inhibitors that exert their effects on apoprotein part of the enzyme
1. Anti enzymes.
2. Inhibitors that denature proteins.
3. Chemical group inhibitors (enzyme poison).
76
Anti enzymes
1. Enzymes that inhibit another enzymes.
2. As anti-thrombin that inhibits thrombin (inhibits coagulation) and activated by heparin.
77
Inhibitors that denature proteins
1. Strong acids.
2. Alkalis.
3. Alcohols.
4. Salts of heavy metals.
78
Chemical group inhibitors (enzyme poison)
1. SH (sulfhydryl) group group inhibitors.
2. OH (hydroxyl) group inhibitors.
79
SH (sulfhydryl) group inhibitors
1. SH group is commonly found at the active site of many enzymes.
2. SH group inhibitors include:
- Oxidixing agents.
- Salts of heavy metals.
80
SH oxidizing agents
Oxidizes the SH group into
81
SH oxidized agents
1. Oxidizes the SH group into disulfide (S-S).
2. H2O2.
3. 2SH + {o} = S-S + H2O
82
SH Salts of heavy metals
1. As HgCl2 (mercury chloride).
2. The positively charged heavy metals binds to the negatively charged sulfur.
3. 2 SH + HgCl2 = S-Hg-S + 2HCl
83
OH hydroxyl group inhibitors
OH group is commonly found in active sites of enzymes.
84
Examples of OH (hydroxyl) group inhibitors
Aspirin: produces acetylation of OH serine of cyclo-oxygenase (COX).
1. Leads to decrease in prostaglandins.
2. Anti-inflammatory and antipyretic action of aspirin.
85
How is enzyme activity regulated?
Regulation of enzymes is achieved by two mechanisms:
1. Changing the amount of the enzymes.
2. Changing the activity of the enzymes.
3 Covalent modification.
86
Changing the amount of the enzymes
The amount of enzymes is controlled by the rate of:
1. Enzyme synthesis.
2. Enzyme degradation.
87
Changing the amount of the enzymes: enzyme synthesis
1. Increase in synthesis: by increase in gene expression of the enzyme (induction) by induced, which may be substrate of the enzyme or hormones.
2. Decrease in synthesis: by decrease in gene expression of the enzyme (repression) by repressor, which may be the product or hormone.
88
Changing the amount of the enzymes: enzyme degradation
By controlling the synthesis or the activity of the enzymes responsible for degradation.
89
Changing the activity of the enzyme
1. Activation of zymogens (proenzymes).
2. Allosteric modifiers (inhibitors and activators).
90
Changing the activity of the enzyme: activation of zymogens (proenzymes)
They are enzymes secreted in an inactive form.
1. Active site is masked by a polypeptide chain and activation occurs by the removal of these polypeptide chains. The active form of the enzyme can the activate its zymogen (autocatalysis or autoactivation).
91
Changing the activity of the enzyme: activation of zymogens (proenzymes) examples
1. Digestive enzymes and enzymes of blood clotting factors (activated by proteases).
2. Pepsinogen and pepsin activated by HCL.
92
Changing the activity of the enzyme: alllosteric modifiers (inhibitors and activators)
Allosteric activators binds to the Allosteric site and makes conformational changes in the active site. It becomes more suitable to bind with the substrate, which increases affinity.
Example: AMP is the Allosteric activator of PFK1.
93
Changing the activity of the enzyme: alllosteric modifiers (inhibitors and activators) effect on Km and Vmax
1. Km: decreased.
2. Vmax: increased.
94
Changing the activity of the enzyme: alllosteric modifiers (inhibitors and activators) effect on Km and Vmax
1. Km: decreased.
2. Vmax: increased.
95
Covalent modification
1. Activation and inhibition by phosphorylation and dephosphorylation.
2. Many enzymes are activated or inhibited by phosphorylation or dephosphorylation.
3. The enzyme is present in 2 interconvertible forms (phosphorylated and dephosphorylated).
4. Phosphorylation occurs by kinase.
5. Dephosphorylation occurs by phosphate.
6. Phosphate is attached to OH of Serine , threonine, and tyrosine.
96
Isoenzymes (isozymes)
Enzymes that catalysts the same reactions but differ in structure, properties, and rate of chemical reactions.
97
Isoenzymes properties
PECCKA
1. Different polypeptide chains.
2. Different electrophoretic mobility.
3. Different clinical uses.
4. Present in the same or different cells.
5. Different Km.
6. Affected in a different way by activators and inhibitors.
98
Isoenzymes examples
1. Lactate dehydrogenase (LDH)
2. Creatine Kinase (CK)
99
Lactate dehydrogenase (LDH)
Characterized by:
- Has 4 polypeptide chains (tetrameric) either H or M.
- 5 Isoenzymes:
1. LDH1: HHHH : present in the heart and increase myocardial infarction (MI).
2. LDH 2: HHHM
3. LDH 3: HHMM
4. LDH 4: HMMM
5. LDH 5: MMMM: present in muscles and liver and increase muscle and liver diseases.
100
Creatine Kinase
- Formed of 2 polypeptide chains (dimer) either B or M.
- 3 Isoenzymes:
1. CK1: BB: In brain and increase brain infarction.
2. CK2:BM: in heart and increase myocardial infarction (MI).
3. CK3:MM: in skeletal muscle more than cardiac muscle and increase muscle diseases.
101
Enzymes
ALAA CLT
1. Amylase
2. Lactate dehydrogenase (LDH)
3. Acid phosphatase
4. Alkaline phosphatase
5. Creatine kinase (CK)
6. Lipase
7. Transaminases:
- Alanine transaminase (ALT)
- Aspartate transaminase (AST)
102
Amylase source
1. Salivary glands.
2. Pancreas.
103
Amylase clinical application
1. Parotitis
2. Pancreatitis
104
Lactate dehydrogenase (LDH) source
1. Skeletal muscle
2. Heart
3. Liver
105
Lactate dehydrogenase (LDH) clinical application
1. Muscle and liver diseases.
2. Hemolysis
3. Tumor marker
106
Acid phosphatase sources
1. Prostate
2. RBC’s
107
Acid phosphatase clinical application
Cancer prostate
108
Alkaline phosphatase sources
1. Liver
2. Bone
3. Intestine
109
Alkaline phosphatase clinical phosphatase
1. Hepatobiliary
2. Bone diseases
110
Creatine kinase (CK) sources
1. Skeletal muscles
2. Heart
111
Creatine kinase (CK) clinical application
1. Muscle diseases.
2. Myocardial infarction.
112
Lipase source
Pancreas
113
Lipase clinical application
Pancreatitis
114
Alanine transaminase (ALT) sources
Liver
115
Aspartate transaminase (AST) sources
1. Liver
2. Heart
116
Alanine (ALT) clinical application
Hepatitis
117
Aspartate (AST) clinical application
1. Hepatitis
2. Myocardial infarction
118
Enzymes for diagnosis of myocardial infarction
1.CK MB
2. LDH1
3. AST
119
Enzymes for diagnosis of pancreatitis
1. Amylase
2. Lipase
120
Enzyme specific for the liver
ALT
121
Enzyme specific for the pancreas
Lipase
122
Classification of Enzymes
IUBMB (international union of biochemistry and molecular biology) developed a system of nomenclature for enzymes.
123
Enzymes are classified into 6 classes
HOTILL
1. Hydrolases: break by addition of water.
2. Oxireductases: oxidation-reduction reaction.
3. Transferases: transfer chemical group from compound to another compound.
4. Isomerases: convert isomer to another isomer.
5. Lyases: break down without addition of water.
6. Ligases: bind 2 compound together.
