C1 - Section 1. INTRODUCTION TO ENZYMES Flashcards
1
Q
is a field of laboratory medicine which focuses on the study of enzymes and their significance to the diagnosis and treatment of diseases.
A
Clinical Enzymology
2
Q
These are substances that catalyzes a given chemical reaction
A
enzymes?
3
Q
The reaction they catalyze are frequently (?) which means which means that they can synthesize and decompose molecules
A
reversible
4
Q
They are (?) types of protein in terms of both structure and function
A
complicated
5
Q
They easily (?) with varying molecular weight and mass
A
denatured
6
Q
These enzymes are (?) which are capable of ionizing either as acid or base
A
amphoteric
7
Q
They are synthesized in an (?) and operates in the presence of a (?)
A
inactive state
cofactor
8
Q
Enzymes are found in all body tissues, they appear in the serum following cellular injury or they may come from degraded cells thus changes in (?) reflects changes in state of health.
A
enzyme concentration
9
Q
non-protein organic biochemical that takes part in the enzyme reaction
A
Coenzymes
10
Q
Essential to the catalytic activity as a CO-SUBSTRATE
A
Coenzymes
11
Q
Diffusible, heat stable, low molecular weight that when combined tightly to enzymes, the coenzyme will be called Prosthetic group
A
Coenzymes
12
Q
Coenzymes E.g.
A
NAD, Pyridoxal phosphate
13
Q
- Inorganic ionic cofactor
A
Activators
14
Q
increase the catalytic activity of an enzyme when it binds to specific site
A
Activators
15
Q
Metabolic regulator of enzyme reaction
A
Activators
16
Q
Usually metal ions (esp. divalent cations)
A
Activators
17
Q
Activators E.g.
A
Mg++, Na+, K+, Zn++
18
Q
- the combined enzyme & coenzyme
A
Holoenzyme
19
Q
- Enzyme without a cofactor
A
Apoenzyme
20
Q
- A coenzyme that cannot be removed from its attachment to an enzyme using dialysis
A
Prosthetic Group
21
Q
Prosthetic Group E.g.
A
Pyridoxal phosphate in transaminase reaction
22
Q
- Substance acted upon by an enzyme & is converted into a new substance
A
Substrate
23
Q
- Substance derived from a transformed substrate
A
Product
24
Q
– Site where substrate interacts with enzymes
A
Active site
25
– Site other than the active site that may lead to either attachment of substrate to the enzyme’s active site or inhibition of attachment
Allosteric site
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– different form of an enzyme with different genetic origins but catalyze the same reaction
Isoenzymes
27
– Results when an enzyme is subject to different post-transitional modification
Isoforms
28
- Refers to the sequence of amino acids joined by peptide bonds to form a polypeptide chain
a. Primary Structure
29
- Conformation of the segments of polypeptide chain
b. Secondary Structure
30
Made up of alpha helices or beta-pleated sheets which are maintained by hydrogen bonds
b. Secondary Structure
31
- Arises from the interactions among side chains/groups of the polypeptide chain
c. Tertiary Structure
32
Structure are bent and folded and maintained by covalent disulfide bond
c. Tertiary Structure
33
- Separate bended & folded structures are put together to form a functional unit
d. Quarternary Structure
34
Enzyme variants –
LDH, Creatine kinase
35
The enzyme action model
a. Enzymes act through formation of (?)
b. The substrate must be bound to the (?)
c. The (?) will then break down to give the reaction products and free the enzyme
d. All enzyme reactions are in theory (?) however, in practice, reactions are usually more rapid in one direction than the other.
enzyme substrate complex
active site of the enzyme
enzyme-substrate complex
reversible
36
refers to the active site being complementary in shape & size to the substrate
37
First presented by Emil Fisher, the lock represents an enzyme and the key represents a substrate.
Lock and Key Theory
38
It is assumed that both the enzyme and substrate have fixed conformations that lead to an easy fit.
Lock and Key Theory
39
Because the enzyme and the substrate are at a close distance with weak attraction, the substrate must need a matching shape and fit to join together.
Lock and Key Theory
40
At the active sites, the enzyme has a specific geometric shape and orientation that a complementary substrate fits into perfectly.
Lock and Key Theory
41
Factors that influence the enzymatic reaction
42
– The rate of enzymatic reaction
Time
43
If the catalytic activity of an enzyme on a substrate is fast, this will mean a shorter reaction time thus liberating the enzyme to act again on the remaining substrate
Time
44
– Commonness between the enzyme and the substrate
Molecular compatibility
45
– Number of substrate that can be reacted
Space availability
46
- capacity of enzymes to recognize and bind only one or few molecules among others
Specificity
47
– when an enzyme can act and catalyze one unique reaction
Absolute specificity
48
– when some enzymes act on different substrates belonging to the same group
Group specificity
49
– an enzyme acts only on the specific isomer
Stereoisomeric
50
o Enzyme conc. is fixed; Substrate conc. is varied
First order Kinetics
51
o Rate of reaction is almost directly proportional to substrate conc. at low values
First order Kinetics
52
o At low concentration of the substrate, only a fraction of the enzyme is associated with the substrate
First order Kinetics
53
o The rate observed reflects the low concentration of the ES complex
First order Kinetics
54
o When maximum velocity is reached, the rate of increase in velocity is “O”
Zero order Kinetics
55
o Reaction rate is unaffected by increased substrate concentration
Zero order Kinetics
56
o Dependent on enzyme concentration
Zero order Kinetics
57
o In this reaction, the entire enzyme is bound to substrate and a much higher rate of reaction is obtained
Zero order Kinetics
58
o Because the entire enzyme is present in the form of the complex, there is now no further increase in ES complex conc. No further increment in reaction rate are possible
Zero order Kinetics
59
- shows the relationship of the reaction velocity to the substrate concentration
Michaelis-Menten Curve
60
- °T considered favorable for enzyme activity (30-37°C or 37 – 40°C)
Optimum temperature
61
– reaction rate is doubled for every 10°C increase
Q10 value
62
: enzyme undergoes inactivation and denaturation
50 – 60°C
63
- the point at w/c the reaction rate is greatest
Optimum pH
64
, many enzymes show maximum activity
At pH 7.0 – 8.0
65
pH value are seen as
low as 1.5 and as high as 10.5
66
may be different in forward and reverse reaction
Optimal pH
67
is important: it affects the three dimensional confirmation of the enzyme
Maintaining pH
68
– increased reaction rate
Activators
69
Bind the substrate to the active site by forming ionic bridges
Activators
70
Orients the substrate so it is attached to the enzyme in the correct configuration
Activators
71
- Decrease the rate of enzyme reaction
Inhibitors
72
- Inhibitors Binds to the active site, blocks access of the S to the E
Competitive inhibition
73
is a structural analog of the substrate, but it is not identical thus breakdown to products do not take place o
competitive inhibitor
74
If substrate conc. Is significantly higher than the inhibitor, the inhibition may be reversible
Competitive inhibition
75
- Binds elsewhere on the E causing change in shape that interferes w/ S binding
Non-competitive inhibition
76
Attachment of the inhibitor to the enzyme does not alter the affinity but the presence of ESI prohibit the formation of products
Non-competitive inhibition
77
- Inhibition-inhibitors binds to the ES complex, if substrate will be increased, there will be increase in ES conc. increasing inhibition, this inhibition does not yield product.
Uncompetitive inhibition
78
- Inhibitors are possible removed from the system; enzyme is fully restored
Reversible inhibition
79
- Inhibitors covalently combine w/ the enzyme
Irreversible inhibition
80
o Physical methods are ineffective in separating inhibitors from the enzymes
Irreversible inhibition
81
o Physical processes that remove inhibitors: dialysis, gel filtration
Reversible inhibition
82
Methods of Enzyme Assay
83
The reactants are combined
Fixed Timed Assays
84
Reaction proceeds for a designated time & is stopped (by inactivating the enzyme)
Fixed Timed Assays
85
reaction is stopped by a weak acid, then measurement is made of the amount of reaction that has occurred
Fixed Timed Assays
86
The reaction is assumed to be linear over the reaction time, the larger the reaction, the more enzyme is present
Fixed Timed Assays
87
Multiple measurements are made at specific time intervals or by a continuous-recording spectrophotometer
Continuous-monitoring or kinetic assay
88
Advantage: Linearity of the reaction is adequately verified
Continuous-monitoring or kinetic assay
89
This is preferred because any deviation in linearity is readily observable
Continuous-monitoring or kinetic assay
90
Proposed by the Commission on Enzymes (IUB)
IU – International Unit
91
Used to standardize the system or reporting of quantitative results
IU – International Unit
92
is the amount of enzyme that will catalyze the reaction of 1 µmol of substrate per minute under specified conditions of temperature, pH, substrate and activators.
IU – International Unit
93
Expressed in terms of U/L or mU/L
IU – International Unit
94
unit of enzyme activity w/c converts 1 mol of substrate per second
Katal
95
conforms w/ the Systemè International (SI) scheme of units
Katal
96
Mole is the unit for substrate concentration while the unit of time is second
Katal
97
Enzyme concentration is then expressed as
katals per liter
98
Factors that influence rate of entry
99
Impaired energy production: promote deterioration of cell membrane
Leakage of enzymes from cells
100
Direct attack on the cell membranes (viruses or organic chemicals)
Leakage of enzymes from cells
101
Reduction in the supply of oxygenated blood perfusing any tissue (e.g. MI)
Leakage of enzymes from cells
102
Genetic deficiency of enzyme production
Altered enzyme production decrease
103
Enzyme production is depressed as a result of disease
Altered enzyme production decrease
104
– NOT a major route for elimination
Urinary excretion
105
Except: Amylase = ↑blood levels (e.g. Acute pancreatitis)
Urinary excretion
106
Inactivated enzymes are removed by the RES
Enzyme Inactivation in Plasma
107
Half – life in plasma: 24 – 48 hours
Enzyme Inactivation in Plasma
108
the enzyme changes in shape during binding to accommodate the substrate
Induced fit Model
109
The induced-fit model is generally considered the more correct version
Induced fit Model
110
This theory maintains that the active site and the substrate are, initially, not perfect matches for each other.
Induced fit Model
111
inorganic activators existing as part of the enzyme molecules
Metalloenzyme
112
Examples of Inhibitors
1. Excess substrate
2. Product of reaction
3. E-S complex does not break to yield products
4. Chemical substances
113
- causes competition between substrate molecules for a single binding site
1. Excess substrate
114
-may be an inhibitor of the forward reaction
2. Product of reaction
115
Disruption of the 3-dimensional structure of the enzyme molecule
ENZYME DENATURATION
116
ENZYME DENATURATION
• May be reversed if:
•denaturation is not extensive
•denaturing agent is removed
117
DENATURING CONDITIONS
1. Elevated temperature
2. Extremes in pH
3. Radiation
4. Frothing
5. Strong salt solution
6. Mechanical trauma
7. Chemicals
118
The (?) is directly proportional to the (?) present in the system
rate of an enzyme-catalyzed reaction
amount of active enzyme
119
SOURCES OF ERRORS IN ENZYME ASSAY
1. Use of Plasma
2. Hemolysis
3. Turbid/Lactassent Serum (Lipemia)
4. Heat labile enzyme
5. Contaminants
120
- Least preferred specimen
- inhibitory effects of anticoagulants on enzyme activity
Plasma
121
Release of intracellular enzyme
Hemolysis
122
- Inhibits CK & Amylase
Turbid/Lactassent Serum (Lipemia)
