Introduction in enzymes Flashcards
Enzymes are mainly
Enzymes are mainly proteins that facilitate biochemical reactions
Ribozyme is an non protein enzyme= RNA splicing
Enyzmes are biological what
catalysts.
Systemic enzymes
Active throughout the body
Tissue specific enzymes
Active in a specfic area
Can measure the acitivity of these enzymes to
-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
Cofactor/ coenzyme
1) Protein or non-protein
2) Permanent or temporary
3) Essential factor for the enzymes which
require cofactor
Competitive inhibition
Compete for enzyme active site
Allosteric inhibition
Binds to a seperate site other then the active site and changes the active site shape
Proenzyme
Zymogens- Inactive or less active precursor of enzyme
- Proteolytic modification required to be
activated
Zymogen examples
Angiotensinogen,
trypsinogen, pepsinogen,
chymotrysionogen, prolipase
Enzyme kinetics
-Enzymes speed up reactions
-Enzymes work by converting a substrate into a product via an
enzyme-substrate complex:
E + S–> ES=
+1
ES–> E+S=
-1
ES—> E +P=
+2
E+P–> ES
-2
Enzyme reaction
E+S–> ES—> E+P
Km= formula
(K-1+ K2)/ K1
Km= Dissociation/ Association
Vmax=
Is the reaction rate when the enzyme is fully saturated by substrate
Km=
Is the substrate concentration that is required to make the speed reach 1/2 Vmax
How does the enzyme speed up the reaction rate
1.) Lowering acitivation energy
2.) increasing the rate constant
3. increasing substrate specificity ( or Substrate concentration)
Reaction rate=
K[S]to the X power [S] to the Y power
Rate= [A]^x * [B]^y
Delta G=
Product energy- Reactants energy
negative delta G =
Exergonic or energy releaseing
Activation energy=
The amount of energy required to make a reaction move forward
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
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.
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
What enzymes dont require a pH of 7-8
Alkaline phosphatase, Acid phosphatase, and Pepsin
Creatine + ATP with enzymes Mg2+ and CK—>
P-creatine + ATP + H+
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
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]
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”
IU or U
The amount of enzyme that produces 1μM
of product per minute under standard
conditions
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
Muscle enzymes
CK and LDH
Creatine Kinase
Size
Catalyzes what
(CK/CPK)
- 82kD dimeric enzyme
- Catalyzes reversible phosphorylation of Creatine
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
Other inhibitors of CK
Mn2+ , Ca2+ , Cu2+ , iodoacetate, other SH binding
proteins
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+
At pH of 6.8 the CK reaction is
Not favored
Creatine stays
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.
Creatine kinase Diseases
- MI
- Rhabdomyolysis
- Muscular dystrophy
CK isomers subunits
Subunits: M & B (40 kD each)
Combination of subunits:
Three CK isomers
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)
CK number one
CK-BB
CK number two
CK-MB
CK number three
CK-MM
Electrophoresis of CK
- electrode
CK-MM
CK-MB
CK-BB
+ electrode
Both subunits ( M and B) in CK have
Both Subunits (M&B) Have Lys residue @ C-terminu
Lysine in M subunit in CK can
Lysine in M subunits can be hydrolyzed by “carboxypeptidase
2 CK MM isoforms
2 Lys removed—> CK MM1
1 Lys removed–> CK MM2
CKMB can undergo the
CK MB could undergo same modification: CK MB1 / CKMB2
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
Clinical Significance of CK
what types of injury
Muscle Injury, Inflammation, Necrosis of SM and CM
“ALL Type of Muscular Dystrophy
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
Progressive Muscular Dystrophy
X chromosome disease
1.**Duchenne Sex-Linked MD (x chromosome)
* 50-100 x URL
* Asymptomatic Female Carrier; 3-6x increased CK
Progressive muscular Dystrophy
Severe what
2.Severe Physical Crush Injury
* 200x URL
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
3 days after Crush injury if the CK value is more then 5000U/L then
higher chance of developing Acute renal failure
Measurement of CK
Creatine + ATP with enzymes Mg2+ and CK —-> Creatine monophosphate + ADP + H+
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
CK sources of errors
- Hemolysis,
- Exercise
increased 10 U/L per Hb (1g/L), due to RBC AK
CK reference range
- Female: 15-171 IU/L
- Male: 46-180 IU/L
- CK-MB: less than 6% of total CK
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
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
LDH cofactors
Zn2+
LDH pH of 8.8-9.8
Lactate + NAD—-> pyruvate + NADH
LDH pH of 7.4-7.8
Pyruvate + NADH—-> lactate + NAD
LDH MW
Composed of
- MW: 134 kD
- Composed of 4 peptide chains of two type: M (or A) & H (or B)
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
Other LDH isomers
- LD-X (LD-C): four X (or C) subunits
- LD-6: from severely ill patients
LDH significance
- Invariably found only in the cytoplasm of the cell
- Hepatic, Cardiac, Skeletal muscle, Hematological Disorders
➡Significantly increased
LD 1 and 2
Heart, Kidney, RBC
LD3
Lung, Spleen, Lymph node, WBC, PLT
LD4 and 5
Skeletal muscle, Liver
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
Acute myoglobulin Infarction markers
Myoglobin, Troponin I &T, CK-MB,
Congestive heart failure marker
BNP
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
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
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
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
