Introduction in enzymes Flashcards
1
Q
Enzymes are mainly
A
Enzymes are mainly proteins that facilitate biochemical reactions
Ribozyme is an non protein enzyme= RNA splicing
2
Q
Enyzmes are biological what
A
catalysts.
3
Q
Systemic enzymes
A
Active throughout the body
4
Q
Tissue specific enzymes
A
Active in a specfic area
5
Q
Can measure the acitivity of these enzymes to
A
-Can measure the ACTIVITY of these enzymes in the blood to
ascertain whether these organs have been or are being damaged.
-Abnormal serum enzyme levels are found in various diseases and
inflammation
6
Q
Cofactor/ coenzyme
A
1) Protein or non-protein
2) Permanent or temporary
3) Essential factor for the enzymes which
require cofactor
7
Q
Competitive inhibition
A
Compete for enzyme active site
8
Q
Allosteric inhibition
A
Binds to a seperate site other then the active site and changes the active site shape
9
Q
Proenzyme
A
Zymogens- Inactive or less active precursor of enzyme
- Proteolytic modification required to be
activated
10
Q
Zymogen examples
A
Angiotensinogen,
trypsinogen, pepsinogen,
chymotrysionogen, prolipase
11
Q
Enzyme kinetics
A
-Enzymes speed up reactions
-Enzymes work by converting a substrate into a product via an
enzyme-substrate complex:
12
Q
E + S–> ES=
A
+1
13
Q
ES–> E+S=
A
-1
14
Q
ES—> E +P=
A
+2
15
Q
E+P–> ES
A
-2
16
Q
Enzyme reaction
A
E+S–> ES—> E+P
17
Q
Km= formula
A
(K-1+ K2)/ K1
Km= Dissociation/ Association
18
Q
Vmax=
A
Is the reaction rate when the enzyme is fully saturated by substrate
19
Q
Km=
A
Is the substrate concentration that is required to make the speed reach 1/2 Vmax
20
Q
How does the enzyme speed up the reaction rate
A
1.) Lowering acitivation energy
2.) increasing the rate constant
3. increasing substrate specificity ( or Substrate concentration)
21
Q
Reaction rate=
A
K[S]to the X power [S] to the Y power
Rate= [A]^x * [B]^y
22
Q
Delta G=
A
Product energy- Reactants energy
23
Q
negative delta G =
A
Exergonic or energy releaseing
24
Q
Activation energy=
A
The amount of energy required to make a reaction move forward
25
Factors influencing the rate of the reaction
what concentrations
- Enzyme concentration
[Enzyme], the faster the product formation
b/c more enzymes = more enzyme/substrate complexes
Substrate concentration
* Substrate readily binds to the enzyme at low concentrations.
* [substrate], the rate of reaction increases.
* But [substrate] too high, enzyme Saturation
26
The shape of a protein effects its
The shape of the protein affects its activity”
Anything that alters the conformation of the
protein/enzyme will have an impact on its activity.
27
pH effects what
* Fluctuation of pH: alter “ionizable group(s)” on the enzyme affect
the enzymes shape.
**significant change in pH can cause the enzyme to denature.
* Change equilibrium position [H+ involved reaction]
* Enzymes require a pH: 7 – 8
* Exceptions: alkaline phosphatase, Acid phosphatase, Pepsin
28
What enzymes dont require a pH of 7-8
Alkaline phosphatase, Acid phosphatase, and Pepsin
29
Creatine + ATP with enzymes Mg2+ and CK--->
P-creatine + ATP + H+
30
Temperate and inhibitors effects what
-Temperature increases increase in molecular collisions increase
in the rate of reaction.
**Too high temperature cause the protein denature decreasing the rate
of reaction.
* Optimal temperature is close to physiological temperature (37ºC)
4.Temperature
5.Presence of Inhibitor influences rate of reactions
31
Single (end) point assays
* Incubate sample with substrate for a period of time
* Measure the end absorbance (O.D.)
* Calculate enzyme level by comparing to the [STD]
32
Kinetic assays
* Incubate sample with substrate
* Measure the absorbance over time at certain increments
* An average change in absorbance (product formation) is
used to calculate “enzyme activity”
33
IU or U
The amount of enzyme that produces 1μM
of product per minute under standard
conditions
34
using data
-Calculate change in absorbance per minute
-Correct for dilution factor and serum volume
-Use molar absorptivity to convert absorbance to μM
-Δ absorbance / molar absorptivity x dilution factor
total volume / sample volume
35
Muscle enzymes
CK and LDH
36
Creatine Kinase
Size
Catalyzes what
(CK/CPK)
* 82kD dimeric enzyme
* Catalyzes reversible phosphorylation of Creatine
37
Creatine kinase cofactor
Active site of enzyme
CK can be partially restored by
* Mg 2+ is a important cofactor [too much Mg 2+ inhibit
the CK ]
➡ CK can be partially restored by treating with
antioxidant such as GSH & NAC
38
Other inhibitors of CK
Mn2+ , Ca2+ , Cu2+ , iodoacetate, other SH binding
proteins
39
At pH of 9.0 Alkaline the reaction is
favored reaction
the substrates Phosphocreatine and ADP are favored
Creatine with enzyme Mg2+ and CK and ATP -----> P-creatine + H+
40
At pH of 6.8 the CK reaction is
Not favored
Creatine stays
41
Creatine Kinase highest activity
Highest activity in the MUSCLE: skeletal & cardiac
(2500 U/g tissue) in skeletal (550 U/g tissue) in cardiac
cf. Also in the brain, kidneys, liver & GI tract.
42
Creatine kinase Diseases
* MI
* Rhabdomyolysis
* Muscular dystrophy
43
CK isomers subunits
Subunits: M & B (40 kD each)
Combination of subunits:
Three CK isomers
44
The three CK isomers
CK-MM (skeletal & cardiac) 98% in SK
CK-MB (Cardiac) 25-30 in Myocardium but CKMM is 70 % of myocardium
CK-BB (Brain)
45
CK number one
CK-BB
46
CK number two
CK-MB
47
CK number three
CK-MM
48
Electrophoresis of CK
- electrode
CK-MM
CK-MB
CK-BB
+ electrode
49
Both subunits ( M and B) in CK have
Both Subunits (M&B) Have Lys residue @ C-terminu
50
Lysine in M subunit in CK can
Lysine in M subunits can be hydrolyzed by “carboxypeptidase
51
2 CK MM isoforms
2 Lys removed---> CK MM1
1 Lys removed--> CK MM2
52
CKMB can undergo the
CK MB could undergo same modification: CK MB1 / CKMB2
53
Other CK isomers
* CK-mt: CK in between inner and outer mitochondrial membrane
Other CK isomers
* CK in macroform: macro CK
* Up to 6% of hospitalized patients’ sera
* Type I (CK (CK-BB) + Immunoglobulin (IgG); Type II oligomeric CK-m
54
Clinical Significance of CK
what types of injury
Muscle Injury, Inflammation, Necrosis of SM and CM
“ALL Type of Muscular Dystrophy
55
CK significance
Progressive Musular Dystrophy
highest in infancy and childhood
Female: 15 – 171 IU/L
Male: 46 – 180 IU/L
1.**Duchenne Sex-Linked MD (x chromosome)
* 50-100 x URL
* Asymptomatic Female Carrier; 3-6x increased CK
2.Severe Physical Crush Injury
* 200x URL
56
Progressive Muscular Dystrophy
X chromosome disease
1.**Duchenne Sex-Linked MD (x chromosome)
* 50-100 x URL
* Asymptomatic Female Carrier; 3-6x increased CK
57
Progressive muscular Dystrophy
Severe what
2.Severe Physical Crush Injury
* 200x URL
58
Drugs can effect what and cause
3.Drugs (pharmacological dose) that could increase serum CK activity
* Statins, Fibrates, Antiretroviral, Ang II Receptor antagonist
Progressive muscular dystrophy
59
3 days after Crush injury if the CK value is more then 5000U/L then
higher chance of developing Acute renal failure
60
Measurement of CK
Creatine + ATP with enzymes Mg2+ and CK ----> Creatine monophosphate + ADP + H+
61
CK reaction is based on the
Oliver- Rosalki method
Creatine monophosphate + ADP with CK enzyme-----> Creatine + ATP
PH of 6.8
ATP + Glucose with HK enzyme----> Glucose-6-P + ADP
Glucose-6-P + NADP+ with G6PDH enzyme -----> NADPH + 6-phosphogluconate
HK = Hexokinase
G6PD = glucose-6-phosphate dehydrogenase
62
CK sources of errors
- Hemolysis,
* Exercise
increased 10 U/L per Hb (1g/L), due to RBC AK
63
CK reference range
* Female: 15-171 IU/L
* Male: 46-180 IU/L
* CK-MB: less than 6% of total CK
64
Lactate Dehydrogenase
Is what reaction
* Reversible reaction * Reaction equilibrium favors the reduction of pyruvate
to lactate @ physiological pH
* Increased pH will favor the oxidation of lactate to
pyruvate
65
Too much Pyruvate or Lactate will inhibit
EDTA inhibits
* Too much pyruvate or lactate inhibits LD activity
Zn2+
* EDTA inhibits LDH activity by binding Zn2
66
LDH cofactors
Zn2+
67
LDH pH of 8.8-9.8
Lactate + NAD----> pyruvate + NADH
68
LDH pH of 7.4-7.8
Pyruvate + NADH----> lactate + NAD
69
LDH MW
Composed of
* MW: 134 kD
* Composed of 4 peptide chains of two type: M (or A) & H (or B)
70
LDH subunits
* LD1 (HHHH; H4)
* LD2 (HHHM; H3M1)
* LD3 (HHMM; H2M2)
* LD4 (HMMM;H1M3)
* LD5 (MMMM; M4)
In order of decreasing anodal mobility in an alkaline medium
So LDH1 is closest to positive electrode and
LDH5 is closest to negative electrode
71
Other LDH isomers
* LD-X (LD-C): four X (or C) subunits
* LD-6: from severely ill patients
72
LDH significance
* Invariably found only in the cytoplasm of the cell
* Hepatic, Cardiac, Skeletal muscle, Hematological Disorders
➡Significantly increased
73
LD 1 and 2
Heart, Kidney, RBC
74
LD3
Lung, Spleen, Lymph node, WBC, PLT
75
LD4 and 5
Skeletal muscle, Liver
76
Quantification of total LD activity:
Monitor NADH at 340 nm
* Serum is preferred sample [should be stored at “room temperature”
cf. Plasma sample: more chance for PLT contamination
LD4 & 5 are labile at cold temperature
* Hemolytic sample (X)
cf. 4000 times more LD in RBC than serum
* EP separation for separation of LD isoenzymes
77
Acute myoglobulin Infarction markers
Myoglobin, Troponin I &T, CK-MB,
78
Congestive heart failure marker
BNP
79
Elevated LDH
▫ LD not specific to cardiac tissue – found in various other tissues as well.
▫ Increase in serum level from ~12 - 24 h post infarc, peaks after ~ 48-72 h,
gradually returns to normal by ~ 7 – 14 day
80
Elevated CK
▫ CK-MB is heart specific
▫ Rises about 4-8 h after infarct, peaks at 12-24 h, and returns to normal in 2-3
days CKMB index = CKMB activity/Total CK activity x 100
CKMB: <6% total CK
81
Elevated Troponins
* Troponins are proteins involved in muscle contraction.
* TnI, TnT, TnC: found in skeletal/cardiac muscle
* Screen for cTnI and cTnT
* Following AMI, levels begin to rise ~3 – 6 h, reach peak levels in 14-24 h, return to
normal in 5 – 10 days
82
Elevated myoglobulin
▫ For early detection (leaks 1-3 h of onset) of AMI.
▫ Peak is reached 5 – 12 hours.
▫ Myoglobin is a small molecule (kidney can freely filter) and thus returns to
normal in 18 – 30 h after the AMI.
▫ Problem: NOT specific
- Present in all muscle cells so non-specific
