ENZYMES INTRO/CK/LDH Flashcards
Enzymes
— _______; is a biological substance that catalyzed a reaction [make the chemical reaction faster]
When the enzymes are absent, the reaction would continue but _______.
— Location of enzymes: within the cells [______, ______,intestines and stomach]
Cellular injury/degradation → the enzymes would be ?
- Detection of _____ when certain enzymes are high.
biocatalyst
slower
saliva
pancreas
released into circulation
disorder
Essential to Physiologic Functioning:
✔ Hydration of _____—> ____ maintenance of the blood. as hydrogen can combine with it as acidic/ basic substances.
✔ _______ conduction
✔ ________ contraction
✔ ________ degradation found in _____ tract [______, _____]
✔ _______ use
CO2
pH maintenance
Nerve conduction
Muscle contraction
Nutrient degradation [GI: amylase, lipase]
Energy use
For a fast chemical reaction to occur, there must be the presence of ________[enzymes] + _________.
● _________ site [the substrate]— water-free cavity, where the substance on which the enzyme acts
- ideal environment, such as a slightly [basic/acidic] or [polar/non- polar] environment, for the reaction to occur.
o _________ complex— fast chemical reaction
● _________ site— area other than the active site: water- free
reactants
substrates
Active
basic
non-polar
ES
Allosteric
A chemical reaction may occur spontaneously if the [2] is higher for the reactant than the products.
_________— reactants have enough energy to break their bond and collide to form new bond [bond between the enzyme & substrate]
free energy [reactants]
available kinetic energy
Activation Energy
_________ Specificity– [strictest] enzyme + only 1 substrate and catalyzed a single reaction
_________ Specificity– + all substrates containing a particular chemical group
_________ Specificity– other enzymes are specific to chemical bonds [hydrogen bonds]
_________ Specificity + predominantly combine with only 1 optical isomer [mirror image] of a certain compound.
Absolute
Group
Bond
Stereoisomeric
_________: gen. temperature for preservation
_________: long term preservation
_________: for cold-labile enzymes
Examples [2]
_________ temperature– reversible inactivation enzyme
_________= NOT RECO → inactivates the enzymes
-20C
2-8C
RT
LD4,LD5
Cold temperature
Repeated Thawing
Enzymes that increase in terms of hemolysis:
KLAMP
potassium
LDH
AST
ACP
Aldolase
Magnesium
Phosphate/Phosphorus
A lactescence and milky specimen means that there is a [INC/DEC] in concentration, and there is the presence of______.
DEC
chylomicrons
Once the counterpart reactant is saturated, and an additional reactants are applied, what will happen to the reaction?
it will not result to faster reaction
In substrate and enzyme concentration, it follows the hypothesis of _______ “even in low substrate conc→ the substrate can ______with free enzyme”
Michaelis & Menten
readily bind
Enzymatic Reaction can be:
✔ _________– the rxn rate is directly proportional to substrate concentration.
✔ _________– rxn rate depends only on enzyme concentration.
First Order kinetic
Zero Order Kinetic
In First Order Kinetic, there is the addition of more _______ to easily find its counterpart.
In Zaro Order Kinetic, there is the addition of more _______ to easily find its counterpart.
substrate
enzyme
_________Enzymes are protein that carry a net molecular charge
— # physiologic enzymatic reactions occur in the pH range of _________ but some enzymes are active in wider pH ranges than others.
[2 Examples]
pH
7.0-8.0
ACP- acid
ALP- basic
TEMPERATURE
[INC] = [INC/DEC] chemical rate rxn
Temperature coefficient
– In every increase of [#] degree = [#]x increase rate rxn; until, of course, the
________ is denatured.
________– optimum temp.
________— [INC] denaturation rate increases as the temp
increases and is usually [significant/nonsignificant]
________— enzymes inactivation _______temperature— enzymes inactive
- ___________→ prevent activity loss until analysis.
10 degree
2x
protein
37C
40-50%
60-65%
Low
Frozen/Refrigerated
___________ non-protein entities that must bind with enzyme for a rxn to occur
- ___________—made up of inorganic cofactors
— function by alternating the enzyme ___________ [enzyme ___________ change that corresponds to the substrate] for proper substrate binding
—Linking substrate to the enzyme/coenzyme, or undergoing ___________.
✔ Metallic: [5]
✔ Non-metallic: [2]
COFACTORS
Activatiors
spatial configuration
shape
oxidation-reduction
Ca, Fe, Mg, Mn, Zn, K
Br, Cl
___________— made up of organic cofactors
— as 2nd substrates for enzymatic rxn.
▪ ___________ between the enzyme and substrate [↑ ___________ of the rxn]
___________– when bound tightly to the enzyme, coenzymes
o E.g. [3]
Coenzymes
Bridge
velocity
Prosthetic groups
Vit, NAD, NADP
Interfere with the reaction= Enzymatic reactions may not progress normally
Inhibitors
physically bind to the active site of an enzyme and compete with the substrate for the active site.
[same shape with the active site= NO ES complex formed]
Competitive Inhibition
Is competitive inhibition reversible or not? If yes, how?
Reversible
By increasing the substrate concentrate, so the possibility of its binding capacity is higher than the inhibitor.
binds an enzyme at a place other than the active site [allosteric site]= NO ES complex produced= NO RXN CATALYZED
Non-competitive Inhibition
Is Noncompetitive inhibition reversible, irreversible or both? explain.
Both
Reversible, by other substances that can bind other than the inhibitors.
Irreversible, when the inhibitors destroys the active site.
True or False
In NonCompetitive Inhibition, Increasing the substrate concentration would not reverse the reaction as it binds the enzyme independently from the substrate [binds to allosteric site]
True
inhibitor binds to the ES complex.
Uncompetitive Inhibition
2 METHODS IN MEASURING THE ENZYMATIC RXN
— Measure based on their _______ and not the absolute ______.
[2]
activity
value
Fixed-time/End point
Continuous Monitoring/Kinetic Assay
- _____________ – single measurement of sample
—– reactants are combined → rxn proceeds for a designated
_______ → rxn stopped [most enzyme inactivation is by the help of _______]
A measurement is made of the ________ that has occurred
[larger reaction= more enzymes are present]
Fixed time/End point
time
weak acid
amount of reaction
_________________ Assay– multiple measurement of enzyme activity at specific time intervals
Continuous monitoring/Kinetic assay
Continuous Monitoring/Kinetic Assay is preferred because?
It is also the most common deviation where enzyme is so ________ → all ________ is used early in the reaction time.
any deviation from linearity is readily observable
elevated
substrate
results in structural cavities.
Tertiary structure
made up of polypeptide chain twisting
Secondary structure
enzyme contains more than one polypeptide unit: spatial relationships between the subunits
Quarternary structure
Catalyze an oxidation–reduction
reaction between two substrates
Catalyze the transfer of a group other than hydrogen from one substrate to another
Catalyze hydrolysis of various bonds
o Breaks substances with the use of _______.
Oxido-reductase
Transferase
Hydrolase
Catalyze removal of groups from substrates without
hydrolysis; the product contains double bonds
Lyase
Catalyze the interconversion of geometric,
optical, or positional isomers
Isomerase
Catalyze the joining of two substrate molecules,
coupled with breaking of the ____________ bond in ATP or a similar compound
Ligases
pyrophosphate
digit of subclass and sub-subclass
2nd + 3rd
digit of class
1st
digit of the serial number specific to each enzyme in a sub- subclass
4th
Dimer w/ 2 subunits
CK Isoenzymes
Dimer w/ 2 subunits
Enzymes with same catalytic function but DIFF physical PROPERTIES.
CK Isoenzymes
Percentage from the total CK:
CK1/BB-
CK2/MB-
CK3/MM-
1%
<6%
94-100%`
CK that is of :
Brain type: Brain tissue
CK-BB
CK that is hybrid type [muscle + brain]: more on from the _______.
CK-MB/2
heart
CK that is muscle type: mostly ______ muscle [Examples]
CK-MM/3
skeletal
LD1-5
— Associated w/ ATP generation in contractile system and is related with muscular system
Creatine Kinase/Phosphokinase
CK function in the muscle cells, it stores Creatine phosphate that is important in ______ production.
ATP
Creatine Kinase/Phoshphokinase is differentiated by 3 methods:
Electrophoresis
Solubility
Inactivation resistance
CK’s arrangement in electrophoresis.
[from cathode to anode]
Cathode- CK3- CK2- CK1- Anode
— Seldomly found in the plasma
Short half life: 1-5 hrs
CK-BB
_____________
Have a high molecular size → cannot pass through
the x Blood Brain Barrier → Confined to the ______ or ______.
CK-BB
brain
cranium
May be seen in the circulation of neonates as this is not developed fully.
CK-BB
— MOST IMPORTANT part of CK
CK-MB
A tissue that compromises 20% of CK-MB and very little to other tissues
Cardiac tissue
is the only tissue from which CK-MB enters the serum, meaning it is quite specific to the ________ muscle
Myocardium
cardiac
CK-MB as indicator of AMI
Rise= _______
Peak= _________
Normalize= ________
4-8 hrs
12hrs-1day
2-3days
Other cardiac markers aside from CK-MB: [3]
LDH
AST
Troponin
— SERUM MAJOR FRACTION @ Skeletal/Cardiac muscle
CK-MM
CK- MM
[INC]: [3]
Hypothyroidism
IM Injection
Muscle activity
o Hypothyroidism: [INC] ___________ + [DEC] ________ due to slow _________
o Muscle activity: __________
o IM: ___________
membrane permeability
CK clearance
slow metabolism
vigorous exercise
muscle damage
MACRO-CK
– midway between MM AND MB
– related to age and sex: associated to ____________.
2 FORMATION THEORIES:
- CK ____ that is found in IgG
- CK ____ that is bound to
lipoprotein
NO clinical significance
MACRO-CK
age, sex
> 50 yrs old female
BB
MM
________________
— Found before MM.
— Bound to the exterior surface of mitochondrial membrane of [3]
Clinical significance: [3]
Mitochondrial CK [CK-Mi]
mitochondrial
muscle
brain,
liver
severe illness
malignant tumors
cardiac abnormalities
Hypothyroidism, malignant hyperpyrexia, Reye’s syndrome, Vibrio Vulnificus — are clinically significant to what CK?
CK-MM
METHODS USED FOR THE MEASUREMENT OF ISOENZYMES OF CK
[4] [+2 subtypes]
- Electrophoresis
- Ion Exchange Chromatography
- Ab used for CK MB/AMI Diagnosis
- Immunoassay
– Tanzer-Gilbarg Assay
–Oliver- Rosalki Assay
REFERENCE method of CK
Electrophoresis
More SENSITIVE, PRICY > electrophoresis
Problem with the _______:
CK-____ merge with CK-____
CK-____ eluted with CK-____
Ion exchange chromatography
Bad column
BB
MB
MM
MB
__________ inhibits ALL the activity of M subunit.
o _________: 2 Subunits M
- entire activity would be
inhibited
o _________: 1 M subunit
- half of the activity would be inhibited
▪ B is left
▪ CK B Activity X [#] = reflect CK___ activity
o _________: NO M subunits
- not affected; isolated to
the _______
Anti-M antibody
CK3
CK2
2x
CK1
brain
Detects MB reliably with minimal reactivity
Detects enzyme protein rather than activity
Immunoassay
In immunoassay for CK, with RBC hemolysis, it will release __________= resulting to falsely [increased/decreased] values of CK
Adenylate kinase/AK
increased
CK IMMUNOASSAY
Storage:
_____C: CK may last for up to 7 days
_____: CK may last for 1 month
Reference values: Total CK Male: ______U/L
- much muscular >
Female: _____ U/L
-20C
4
15-160
15-130
TANZER-GILBARG ASSAY
[_____pH @340 nm]
[Forward/Direct reaction or Reverse/Indirect reaction]
OLIVER-ROSALKI ASSAY
[_____pH @_____ nm)
[Forward/Direct reaction or Reverse/Indirect reaction]
9.0pH
Forward/Direct
6.8 pH
340
Reverse/Indirect
Reactions in:
Tanzer Gilbarg
Creatine + ATP –[CK]– Creatine phosphate + ADP
ADP + Phoshoenol pyruvate —ATP + Pyruvate
Pyruvate + NADH + H –[LD]– Lactate + NAD
Reactions in:
Oliver Rosalki Assay
Creatine phosphate + ADP –[CK] – Creatine + ATP
ATP + glucose – ADP + G6P
G6P + NADPH– [GDPD]– 6phosphogluconate + NADP
Catalyzes the interconversion of lactic acid and pyruvic acid
o Lactate + ______ —> Pyruvate + ______.
Coenzyme: ________.
Lactate dehydrogenase
NAD
NADH
NAD
Tetrametric molecules containing 4 subunits of 2 possible forms [H-heart, M-Muscle]
LD Isoenzymes
LD HHHH & LD HHHM are indicators for these 2 conditions
heart, RBC
AMI
Intravascular hemolysis
LD HHMM is an indicator for this condition
pulmonary disorders
[pulmonary carcinoma]
LD HMMM is an indicator for this condition
intrahepatic disorder
LD MMMM is an indicator for this condition
muscular dystrophy
Abnormal type of LD Isoenzymes
✔ _____ Band to the electrophoresis
✔[2 conditions] → could lead to ______ damage.
6th
impending death
arteriosclerotic cardiovascular failure
liver
LD isoenzyme designated for liver
LD HMMM/4
LD isoenzyme designated for lungs, lymphocyte, spleen, pancreas
LD HHMM/3
LD isoenzyme designated for the skeletal muscle
LD MMMM/5
LD isoenzyme designated for heart and RBC
LD HHHH/1
LD HHHM/2
LD is NON-SPECIFIC, thus it should be differentiated through their concentration.
[Concentration order from highest to lowest]
- LD 1& 2 have same tissue sources but LD___ has more.
_____________
–Concentration of LD1 becomes higher than LD2.
–Indicates [2]
LD2–LD3–LD1–LD4–LD5
1
Flipped pattern
AMI
Hemolyzed sample
MEASUREMENT OF LD ISOENZYMES
Electrophoresis
Immunoinhibition/Chemical Inhibition
Substrate affinity
DIAGNOSTIC SIGNIFICANCE
LDH is increased in these 6 disorders:
[Highest] in: [2]
- LDH is a ______ marker
Renal disorder
Hepatic disorder
Cardiac disorder
Skeletal disorder
Hematologic disorder
Neoplastic disorder
pernicious
hemolytic disorder AMI
Cardiac
LDH in AMI
- Rise: _____ hrs
- Peak: _____ hrs
- Normalize: _____days
12-24hrs
48-72hrs
10 days
LDH Isoenzymes METHODS [2]
- Wacker
- Wrobleuski and La Due
Reference value of LDH
100- 225 U/L
- Wacker Method [_____ pH @ 340 nm]
- Wrobleuski and La Due [______pH @_____nm]
8.3-8.9 pH
7.1-7.4 pH
340nm
__________________.
✔ 3X faster > to wacker method, [small/large] sample is needed
✔ Susceptible due to [2]
✔ HEMOLYSIS should be prevented because?
o LDH is a ______ labile
use ____ in storage
▪ LD___ is the MOST LABILE.
Wrobleuski and La Due
small
substrate exhaustion
loss of linearity
RBC contains 100-150x the LDH concentration.
cold
RT
5
