ENZYMOLOGY Flashcards
(149 cards)
1
Q
Biologic proteins that catalyze biochemical reactions
A
ENZYME
2
Q
Enzymes are _________ proteins, produced by living cells
A
INTRACELLULAR
3
Q
Enzymes affect the ________ or speed of a chemical rxn;
__________ its chemical reaction
A
VELOCITY, speeding up
4
Q
In an enzymatic process, enzymes are not _______ , ________, ________ nor ________ in composition; it only affects the speed
A
consumed, modified, altered, changed
5
Q
Enzyme action is specific with ________ substrates
A
ORGANIC
6
Q
In the lab, enzymes are measured in terms of their ________ and not in their __________
A
ACTIVITY, absolute values (concentration)
7
Q
Activity of enzyme to a substrate is _________ with its concentration
A
directly proportional
8
Q
Greater Enzymatic Activity =
A
High Enzyme Concentration
9
Q
in serum, enzyme concentration is normally low, if it is high, it is indicative of:
A
a. Cellular Injury/ Damage
b. Increase Membrane Permeability
10
Q
Hastens respiration process
A
Hydration of CO2
(Carbonic Acid = buffers the pH)
11
Q
Transmission of nerve impulses
A
Nerve Induction
12
Q
Helps in movement & heat generation
A
Muscle Contraction
13
Q
Digestive juices contain enzymes to facilitate the interconversion of complex molecules in the diet. Absorption of macromolecules in the small intestine
A
Nutrient Degradation/ Digestion
14
Q
Assist in growth & metabolism of a person
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Growth & Reproduction
15
Q
Conversion of energy stores (in ADIPOSE)
A
Energy Storage & Use
(Creatine Phosphate = ATP for muscles)
16
Q
Components of an Enzyme
A
- ACTIVE SITE
- ALLOSTERIC SITE
17
Q
A waterless cavity of an enzyme where substrates bind and undergo chemical reaction
A
ACTIVE SITE
18
Q
Binding site for SUBSTRATE
A
ACTIVE SITE
19
Q
Specific substrate can only bind to the active site of the enzyme
A
Enzyme-Substrate Specificity
20
Q
A waterless cavity other than the active site that binds regulatory or effector molecules.
A
ALLOSTERIC SITE
21
Q
Activators of Enzymes
A
1- Substrate (lab)
2- Cofactor (in the body)
22
Q
Substances acted upon by enzymes
A
SUBSTRATES
23
Q
Nonprotein substances added in the enzyme substrate complex to manifest an enzyme activity (to allow enzyme reaction)
A
COFACTORS
24
Q
To ENHANCE enzymatic activity
A
COFACTORS
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Organic cofactor that hastens enzymatic reactions
COENZYME (2nd Substrate)
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COENZYME examples
NAD & NADP
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Facilitates enzyme-substrate binding by altering the spatial configuration of the enzyme active site
ACTIVATOR
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ACTIVATOR EXAMPLES
1. Metallic Type: Mg, Ca, Fe, Zn, Mn
2. Non-metallic Type: Cl, Br
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Enzymes with similar enzymatic activity/ reaction but differ in their physical, biochemical, and immunologic characteristics
ISOENZYME
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One way of differentiating/ fractionating isoenzymes is with the use of _____________
electrophoresis.
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In the lab, ___________ is more preferred than _________ measurement due to its _______ to target tissue
isoenzyme measurement, total enzyme activity, HIGH SPECIFICITY
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ISOENZYME EXAMPLES
1. Creatine Kinase (CK1, CK2, CK3)
2. Lactate Dehydrogenase (LDH1 to LDH5)
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Protein portion of the enzyme
APOENZYME (Inactive Enzyme)
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If the apoenzyme is subjected to _________, enzyme activity is lost
denaturation
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Denaturation promotes at ________, would promote destruction of ____________
56degC, tertiary proteins
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When an apoenzyme is tightly bound/attached with a coenzyme, it is termed as a _____________
prosthetic group
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An active substance formed by the combination of a coenzyme and an apoenzyme (the product)
HOLOENZYME (Active Enzyme)
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The naming of enzymes and its classifications was recommended by the
Enzyme Commission or the International Union of Biochemistry
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The LONGER name
Defines the substrate acted on, or the reaction catalyzed, and possibly the coenzyme involved
Systematic Name
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The usable/ trivial name of the enzyme
Frequently used name
Recommended Name
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Always starts with E.C. followed by the 4 digits
1st – class of enzyme
2nd – subclass of enzyme
3rd – sub-subclass of enzyme
4th – serial number specific to enzyme sub-subclass
EC (Enzyme Commission) Numerical Code
42
Always starts with E.C. followed by the 4 digits
1st –
2nd –
3rd –
4th –
EC (Enzyme Commission) Numerical Code
1st – class of enzyme
2nd – subclass of enzyme
3rd – sub-subclass of enzyme
4th – serial number specific to enzyme sub-subclass
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6 1st DIGIT CLASSIFICATION OF ENZYME
1. Oxidoreductases
2. Transferases
3. Hydrolases
4. Lyases
5. Isomerases
6. Ligases
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Catalyzes the reduction oxidation reaction between 2 substrates; loss or gain of electrons; these enzymes utilize NAD/ NADP coenzymes (redox reaction)
Oxidoreductases
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A– + B → A + B–
Oxidoreductases
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In Oxidoreductases, ___ is the commonly transferred atom
H+
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The conversion of the coenzymes NAD and NADP is catalyzed by _______
oxidoreductase
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Catalyzes the transfer of a chemical group such as phosphate, methyl, among others (other than hydrogen) between two substrates
Transferases
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A-X + B → A + B-X
Transferases
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Transferases examples
1.AST/ SGOT (aspartate serum transferase)
2.ALT/ SGPT (alanine aminotransferase)
3.Gamma-glutamyl transferase (GGT)
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Dehydrogenase examples
1. Lactate Dehydrogenase (LDH)
2. Glucose-6 Phosphate Dehydrogenase (G-6-PD)
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Kinases example/s
Creatine Kinase (CK)
- Transfers PHOSPHATE groups
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A–B + H2O → A–OH + B–H
Hydrolases
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Hydrolysis of ether and ester & various bonds
Hydrolases
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In the presence of water, hydrolases can _______ on different chemical bonds; when water is introduced, the bond is _______ (separates substrate A from substrate B).
cleave, cleaved
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There must always be _____ for hydrolases to function
water
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targets glycosidic bond connecting complex sugars
.Amylase
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fats
Lipase
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organic phosphomonoester bond
Phosphatase
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6 Hydrolases examples
1. Amylase
2. Lipase
3. Phosphatase
4. Cholinesterase
5. 5-nucleotidase
6. ACE (acetyl cholinesterase)
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ATP → cAMP + PPi
Lyases
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Removal of groups from substrate without hydrolysis/ presence of water. The product remains having double bonds
Lyases
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cAMP
PPi
cAMP - Cyclic Adenosine Monophosphate
PPi - Intermediate Diphosphate
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Lyases example/s
1.Glutamate Decarboxylase Aldolases
2. Fructose biphosphate aldolase (ALS)
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enzyme
that acts on carbohydrates
Fructose biphosphate aldolase (ALS)
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A → B
Isomerases
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The interconversion of geometric, optical, and positional isomers.
Isomerases
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It would catalyze the interconversion of substrate A to become substrate B
Isomerases
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Ab + C → A–C + b
Ligases
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Joining of two substrate molecules coupled with the breaking of pyrophosphate bond in ATP
Ligases
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Enzymes catalyze physiologic reactions by __________ that the reactants must reach
lowering the activation energy level
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energy required for chemical reactions to proceed; important for product formation
ACTIVATION ENERGY
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The _________ the activation energy required, the ________ the product formation
higher/greater
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The _________ the activation energy required, the ________ the product formation
higher/greater, longer
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The _______ the activation energy required, the ______ the product formation (in presence of enzymes)
lower, faster
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The enzyme reaction is always _______ with the substrate.
reversible
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Enzymes combined with only one substrate and catalyzes only one corresponding reaction
Absolute Specificity
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Enzymes combined with all substrates containing a particular chemical group
Group Specificity
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Enzymes attached to substrates with specific chemical bonds
Bond Specificity
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Enzymes combine with only one specific optical isomer
Stereoisometric Specificity
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Higher Substrate =
More Enzymatic Reaction
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Enzyme is greater than substrate
FIRST ORDER KINETICS (E > S)
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Reaction rate is proportional to substrate conc.
FIRST ORDER KINETICS (E > S)
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Rate depends on concentration of substrate
FIRST ORDER KINETICS (E > S)
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when substrate conc. reaches the maximal value, addition of more substrate could no longer increase the rate of reaction as enzymes are already exhausted
Saturation Kinetics
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Only a fixed/ constant number of substrate (in excess) is converted to product per second
ZERO ORDER KINETICS (S > E) → used in the lab
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In enzymatic reaction, we prefer that substrate is GREATER than enzyme → as long as substrate is in excess, there will be enzyme activity
ZERO ORDER KINETICS (S > E) → used in the lab
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If the enzyme activity or concentration is __________, it cannot be measured by the machine, thus, it must be _________ → retest
too HIGH, serially diluted
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lowers the conc. of enzyme
SERIAL DILUTION
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Reporting of diluted serum
result of diluted serum is not reported, rather multiply the dilution factor to diluted serum
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Higher Enzyme Concentration = More Substrate Binding = Faster Reaction
Enzyme Concentration
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Velocity of reaction is proportional to enzyme as long as there is zero order kinetics
Enzyme Concentration
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Normal except G-6-PD
LOW enzyme conc
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HIGH enzyme conc.
Clinically Significant
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Most physiologic reactions occur between a pH range of ________
7.0-8.0 (close to plasma pH of 7.35-7.45)
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If the pH is too high or too low (extreme pH), enzymes are usually __________ , because enzymes are __________.
denatured/ inactivated, ergo proteins which undergo denaturation when pH is too acidic or too basic
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can survive in extremely low and extremely high pH levels respectively
Acid phosphatase (pH 3-5) and Alkaline phosphatase (pH 9-10)
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Tests for enzymes are done at __________ to mimic the ________ happening in the body.
body temp. 37°C, in vivo reaction
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Assay temperature should be constant within ____ in which the enzyme is active at ____ , ____, and ____.
±0.1, 25°C, 30°C, 37°C
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2 Temperatures for Enzymatic Testing:
1. Room Temperature (25°C) – longer incubation
2. Warm/ Body Temperature (37°C) – shorter; optimum temperature
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Increased Temp.. =
Increased Rate of Chemical Rxn
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Q10
(Temp. Coefficient) = Increased incubation temperature by _____ will have a characteristic of ______ in enzyme activity
10°C, twofold increase
103
Extreme Temp. =
Decreased Enzyme Activity
104
When the temp. exceeds 37°C, the activity starts to _____; which is _____
decrease, irreversible
105
significant decrease in enzyme activity (denaturation of tertiary structure)
40-50°C
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significant decrease in enzyme activity (denaturation of tertiary structure)
40-50°C
107
significant denaturation/ inactivation
56°C
108
proteins are completely inactivated
60-65°C
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Most enzymes are THERMOLABILE (inactive at high temperatures) except
The placental type of Alkaline phosphatase (Regan Alkaphose) which is thermoresistant/
thermostable
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At low/ cold temperatures, enzymes are _______ ; but are preserved and become activated when temperature is ________
reversibly inactive, increased to room/ body temp
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Non protein that enhances the reaction
Cofactors
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It must be present in excess
Cofactors
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when added to the enzyme structure could alter spatial configuration of the enzyme for proper substrate binding
Inorganic/ Activators
114
serves as 2nd substrate for enzymatic reaction (NAD). When coenzymes are increased, the velocity of the enzyme activity could also increase
Organic/ Coenzyme (prosthetic groups):
115
Agents that could interfere with the enzyme-substrate reaction
Inhibitors
116
competes with the substrate on the active site of the enzyme (alters/ slows down velocity by preventing enzyme binding)
Competitive Inhibitors
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it binds on the site (allosteric site) other than the active site
Non-competitive Inhibitors
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binds to enzyme-substrate (ES) complex
Uncompetitive Inhibitors
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Increased Inhibitors =
Decreased Enzymatic Activity
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Storage for substrates & coenzymes (reagents)
2-8degC [refrigerated]
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Storage for storage of LDH (LD4 and LD5 are cold labile)
Room temp. (20-24 degC)
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Storage for longer preservation of enzyme found in samples (serum)
-20degC or colder [freezer]
123
Why is thawing of frozen serum is only done for one time?
Multiple freeze thawing = can destroy enzyme
(denaturation) = falsely decreased
124
Factors that Influence Enzymatic Reactions
1. Substrate Concentration
2. Enzyme Concentration
3. pH
4. Temperature
5. Cofactors
6. Inhibitors
7. Storage
8. Interferences
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It is the energy required to raise all molecules to the transition state in a chemical reaction so that products may be formed
Activation Energy
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Increase in product concentration
= ↑ Rate of Substrate Conversion = ↑ Enzyme Concentration
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Decrease in substrate concentration
= ↑ Products formed = ↓ Substrate Concentration
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Decrease in coenzyme concentration (NADH)
Oxidized NAD (decreased) → reduced NADH (increased absorbance)
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Always performed in zero-order kinetics (w/ fixed substrate)
Catalytic Activity
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Performed during the linear phase of reaction
Measurement of Catalytic Activity
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Simple outright procedure (only 1 measurement)
Fixed Time/Two-Point Assay
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The reagents are combined, the reaction proceeds, the reaction is stopped and the amount of reaction is measured
Fixed Time/Two-Point Assay
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Multiple enzyme activity is included in the procedure
Continuous Monitoring/ Kinetic Assays
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Multiple measurements at specific time intervals or continuous measurement as absorbance changes.
Continuous Monitoring/ Kinetic Assays
134
Deviation from zero kinetics can be observed
Continuous Monitoring/ Kinetic Assays
135
30-60s interval; then absorbance is noted
Continuous Monitoring/ Kinetic Assays
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2 Types of Enzymes Involved in Continuous Monitoring/ Kinetic Assays
1. Primary Enzyme
2. Secondary/ Coupling/ Indicator Enzyme
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target enzyme in serum
Primary Enzyme
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rely on primary enzyme activity for the reaction to proceed
Secondary/ Coupling/ Indicator Enzyme
139
An expression of the relationship between the velocity of the enzymatic reaction and the substrate concentration.
Michaelis-Menten Equation
140
The amount of enzyme that will catalyze the reaction of 1 μmol of substrate per minute (umol/ min)
IU; International Unit (EC) - [μmol/min]
141
Used by the Enzyme Commission
IU; International Unit (EC) - [μmol/min]
142
The amount of enzyme that will catalyze the reaction of 1 mol of substrate per second (mol/s)
Kat; Katal (SI) – [mol/s]
143
Used by the Système international
Kat; Katal (SI) – [mol/s]
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The actual enzyme concentration
Enzyme Mass
145
Enzyme mass can be quantified using _______; enzymes are proteins with charged differences depending on the ___ and the isoelectric point
electrophoresis, pH, isoelectric point
146
2 enzyme theories
1. Emil Fisher’s/ Lock & Key Theory
2. Kochland’s Induced Fit Theory
147
Based on the substrate binding to the active site of the enzyme
Kochland’s Induced Fit Theory
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The shape of the key (the substrate) must fit into the lock (the active site of the enzyme)
Emil Fisher’s/ Lock & Key Theory
