LEC1 - ENZYME INTRO Flashcards
biologic proteins that catalyze biochemical reactions without altering the equilibrium point of the reaction or being consumed or changed in composition
ENZYMES
e same catalytic function but may differ in select physical properties, such as electrophoretic mobility,
solubility, or resistance to inactivation
is generally used when discussing
such forms of the enzymes, although the International Union of Biochemistry (IUB) suggests
restricting this term to multiple forms of similar genetic origin
isoenzyme
An +_____ results when an
enzyme is subject to posttranslational modifications with a functional group added to an amino
acid. Isoenzymes and isoforms contribute to heterogeneity in properties and function of
enzymes because these measured properties are influenced by changes in amino acid
chemistry and the resulting changes in structural features.
isoform
classification of practical or trivial names of enzyme
According to the name of the substrate with the addition of the suffix “ase”
According to the type of reaction they catalyzed.
Transfer of amino group from substrate to another - ____
transferase
Transfer to phosphate group from a high energy phosphate compound to its substrate - _____
kinase
Effect of hydrolysis on phosphate esters – ___
phosphatase
Removal of hydrogen atoms from its substrate - ____
dehydrogenase
systematic name is According to the numerical designation given by the ____
Enzyme Commission (E.C.)
Systematic name for lactate dehydrogenase
E. C. 1. 1. 1. 27
systematic name for amylase
E. C. 3. 2. 1 .1
systematic name for alanine aminotransferase
E. C. 2. 6.1. 2
removal or addition of electrons (reduction-oxidation [“redox”] reaction.
Oxidoreductases
systemic name
discuss how the numbers are given
1st - class
2-3rd - subclass
4th- serial number
classification of Oxidoreductases
oxidase
dehydrogenase
example of oxidase
cytochrome oxidase
example of dehydrogenase
lactate dehydrogenase (LDH)
malate dehydrogenase (MDH)
isocitrate dehydrogenase (ICD)
- catalyze the transfer of a chemical group other than hydrogen from one substrate to another
Transferase
examples of transferase
(a) aspartate aminotransferase (AST)
(b) alanine aminotransferase (ALT)
(c) creatine kinase (CN) or creatine phosphokinase (CPK)
(d) gamma-glutamyl transferase (GGT)
(e) ornithine carbamyl transferase (OCT)
- hydrolyze the splitting of a bond by the addition of water (hydrolysis reaction
Hydrolase
classification of hydrolase
esterases
peptidases
glycosidase
examples of esterases
acid phosphatase (ACP)
alkaline phosphatase (ALP)
cholinesterase (CLS)
lipase (LPS)
example of peptidase
trypsin (PTS)
pepsin (PPS)
leucine aminopeptidase (LAP)
example of glycosidase
amylase (AMS)
amylo 1,6 glycosidase
galactosidases
remove groups from substrate without hydrolysis, leaving only double bonds in the molecular structure of the product.
Lyases
examples of Lyases
(a) aldolases
(b) glutamate decarboxylase
(c) pyruvate decarboxyiase
(d) tryptophan decarboxylase
catalyzes the interconversion of geometric, optical or positional isomers of the substrate compound
isomerases
examples of isomerases
(a) glucose phosphate isomerase
(b) ribose phosphate lsomerase
- joins two substrate molecules together using the energy released from hydrolyzing a pyrophosphate bond to a high-energy phosphate compound.
Ligases (Synthetases)
ligases is Coupled with breaking the ___ bond in ATP
pyrophosphate
example of ligases (synthetases)
Glutathione synthetase
an active substance formed by combination of a coenzyme (cofactor) and apoenzyme.
holoenzyme
formula for holoenzyme
cofactor X apoenzyme
the protein portion subject to denaturation, in which the enzyme loses its activity.
apo enzyme
Catalytically inactive protein when cofactor is removed
apoenzyme
are apo enzyme heat labile and dialyzable.
yes
enzymes present in an individual with similar enzymatic activity but differ in their physical biochemical and immunologic characteristics
isoenzyme
- enzyme whose metal ions are intrinsically part of the molecule such as catalases and cytochrome oxidase
Metalloenzyme
inactive precursor of enzymes,
proenzyme
proenzyme are also referred to as ___
zymogens
substances acted upon by the enzymes which are specific for each of their particular enzyme.
Substrates
these are non-protein substance/compounds needed by an enzyme before enzymatic activity can be manifested. ____ are thermostable and dialyzable
Cofactors
the cofactor in Organic molecule act as ___.
Coenzyme
It hastens enzymatic reaction but undergoes a change or is consumed to another product
cofactor
examples of co factor
NAD – nicotinamide Adenine dinucleotide
NADP - nicotinamide Adenine dinucleotide phosphate
it’s essential to achieve absolute enzymatic activity
cofactors
the cofactor in Metal ion as an ___
Activator
these are inorganic ions which alter the spatial configuration of the enzyme for proper substrate binding
activators
these are inorganic ions attached to molecule
metalloenzymes
a cofactor in metal ion - activator
In such, the metal ion may serve as:
a bridge to hold the substrate and enzyme together
the primary catalytic center
stabilizing agent In the conformation for catalytic activity.
An enzyme (E) catalyses a reaction by combining with its substrate (S) to create an ______
enzyme—substrate complex (ES)
The enzyme-substrate complex according to _____ can either dissociate back to E + S or breakdown to product (P) and free enzyme (provided that the product has a low affinity for the enzyme).
Michaelis and Menten
THE _____ GIVES THE MEANS TO DETERMINE TOTAL ENZYME CONCENTRATION IN SERUM AND OTHER BODY FLUIDS
MICHAELIS-MENTEN EQUATION
Accurately describes virtually all single-substrate enzyme-catalyzed reactions and many bisubstrate reactions in which the concentration of one substrate is constant throughout the course of the reaction.
Michaelis-Menten Equation
Types of Specificity
absolute
group
bond
stereoisomeric
a type of specificity in which the enzymes combine with only one substrate and catalyzes only one corresponding reaction
absolute specificity
a type of specificity in which the enzymes combining with all substrates containing a particular chemical group
group enzymes
a type of specificity in which enzymes are specific to chemical bonds
bond specificity
a type of specificity in which enzymes are predominantly combined with only one optical isomer of a certain compound
stereoisomeric specificity
____’s LOCK and KEY THEORY
Emil Fischer
It is based on the rigid enzyme molecule into which the substrate fits. The shape of the key (substrate) must conform into the lock (enzyme).
Emil Fischer’s LOCK and KEY THEORY
It is based on the attachment of a substrate to the active site of an enzyme, which then causes conformational changes in the enzyme
Koshland’s INDUCED FIT THEORY
This theory Is more acceptable because the protein molecule Is flexible enough to allow conformational changes and also allow some explanation on the influence of hormones on enzymatic activity.
Koshland’s INDUCED FIT THEORY
This phenomenon states that a certain enzyme has the ability to adapt to their biochemical systems
enzyme induction
Factors Affecting Enzyme Reactions:
enzyme concentration
substrate concentration
temperature
hydrogen ion concentration or pH
An increase in the concentration of enzyme produces an increase in the rate of reaction, provided that the other conditions remain the same and that constant but excess amount of substrate Is present
enzyme concentration
Temperature - The rate of any chemical reaction is usually increased 2-3 times for every _______degrees Celsius rise in temperature
I0* C
Enzymatic reactions proceed at their fastest rate at an optimum pH and are considerably slowed or even stopped at higher or lower pH values
Hydrogen Ion Concentration or pH
Types of Reaction Order:
Zero Order Reaction
First Order Reaction
reaction order
is the rate of reaction linear with time, independent of concentration of substrate and directly proportional to enzyme concentration.
Zero Order Reaction
reaction order
the rate of reaction is determined by the concentration of substrate as well as of enzymes (the rate of reaction changes continuously with time as the substrate is consumed
First Order Reaction
- These are substances that compete with the substrate for enzyme binding because they are chemically analogous to the substrate and bind to the active sites of enzymes
Competitive Inhibitor
a type of inhibitor where both substrate and inhibitor compete for the same active site of the enzyme
competitive inhibitor
a type of inhibitor that has the ability to alter the apparent michaelis-menten constant equation
competitive inhibitor
how to increase the rate of reaction if there’s a presence of competitive inhibitor
dilution of the serum
These are substances that do not resemble the substrate and bind to the enzyme in areas other than the active site
Non- competitive Inhibitor
inhibits enzyme by binding to the enzyme-substrate complex
Uncompetitive Inhibition
among the type of inhibition, which one is reversible
only the competitive inhibition, both uncompetitive and non competitive is irreversible
– has the ability to bind to either only the enzyme or the ES complex at a different site from the substrate active site
Mixed Inhibitor
MEANS OF MEASURING ENZYME ACTIVITY
Change in Coenzyme Concentration
Increase in Product Concentration
Decrease in Substrate Concentration
Types of Enzyme Assays
Endpoint Analysis
Multi-point and Kinetic Assay
Use of Coupled Reactions
a type of enzyme assay which the Reaction is initiated by addition of substrate
Endpoint Analysis
a type of enzyme assay which the Reaction is allowed to proceed for a period of time
Endpoint Analysis
a type of enzyme assay which the Measurement is done at the end of the react
Endpoint Analysis
Endpoint Analysis disadvantage
underestimation of the “true” enzyme activity and linearity of reaction cannot be observed
a type of enzyme assay that is about the Change in concentration of the indicator substance at several intervals
Multi-point and Kinetic Assay
a type of enzyme assay that is about the Continuous measurement of change in concentration as function of time
Multi-point and Kinetic Assay
a type of enzyme assays in which Enzymatic activity is measured by coupling the activity with colorimetric reaction
Use of Coupled Reactions
UNITS FOR EXPRESSING ENZYME ACTIVITY
International Unit (I.U. or U)
Katal Unit (K.U.)
Equivalent to the amount of enzyme that catalyzes the conversion of 1 micromole of substrate per minute under controlled conditions.
International Unit (I.U. or U)
Equivalent to the amount of enzyme that catalyzes the conversion of 1 mole of substrate per second under controlled conditions
Katal Unit (K.U.)
1.0 IU = _____nkat
17
PITFALLS IN ENZYME ASSAYS
Hemolysis cause falsely _____ values due to the release of enzymes from red blood cells.
elevated
PITFALLS IN ENZYME ASSAYS
is the preferred specimen due to the adverse effects of anticoagulants on enzyme activity.
Serum
PITFALLS IN ENZYME ASSAYS
Lactescent or milky serum causes variable ___ by the spectrophotometer.
absorption
PITFALLS IN ENZYME ASSAYS aside from hemolysis, use of serum, and lactescent or milky serum
storage ad presence of inhibitors
Storage of Samples
Most enzymes are stable at ____ for at least 24 hrs
6*C
storage of samples
Few enzymes are ___ at refrigerator temp (LD 4 and 5)
inactivated
Quality Control Program for Enzyme Assays
- Strict adherence to zero-order kinetics
- Proportionality with increments of sample
- Use of pooled frozen serum or stable reference materials as controls
- Replicate measurements to evaluate precision of assays
enzymes are active at what temp
25C, 30C, or 37*C
the optimum enzymatic temperature is at 37*C, however, there’s one enzyme that exhibit denaturation at this range
creatine kinase
___ means that for every 10*C increase in temp, there will be 2fold increase enzyme activity
temperature coefficient (Q10)
most physiologic reactions occur in the ph range of
pH 7.0 to pH 8.0
low temperatures will cause __ to enzyme
make it reversibly inactive
repeated freezing and thawing to enzymes causes ___
denaturation of proteins
temp for preservation for a longer period of time
-20*C
ideal storage temp for substrate and coenzymes
2-8C
ideal temp for LDH
room temp (LD4 and LD5)
hemolysis can cause __ to enzyme concentration
increases enzyme concentration
what can lactescense or milky specimen do to enzyme concentration
decrease
it is a water-free cavity where the substrate interacts with a particular charged molecule
active site
it is an amino acid residue with a 3dimensional protein structure
active site
it is a cavity other than the active site
allosteric site
it serves as an attachment site of a non competitive inhibitor
allosteric site
when bound tightly to the enzyme, the coenzyme is called ___
prosthetic group
zymogen is also known as
proenzyme
are enzymes-antibody complexes
macroenzymes
is a model for enzyme kinetics, where the enzymatic reaction is illustrated by the relationship between the rate of the formation of a product and concentration of the substrate
michaelis-menten kinetics
is a kind of reaction wherein one or the other reactant (like a substrate) is altered by increasing or decreasing its concentration
pseudo-first-order reaction
units for expressing enzymatic activity
one micromole of substrate per minute
international unit (IU or U)
units for expressing enzymatic activity
one mole of substrate per seconds
katal unit
this units for expressing enzymatic activity quantified enzymes based on their activity rather than absolute values
katal unit
this units for expressing enzymatic activity uses activity unit to report enzyme levels
katal unit
the preferred specimen for the measurement of enzymes
serum
alternative specimen for the measurement of enzyme is heparin, however, it inhibits what enzyme
AST and CK
these Ag can cause false decreased enzymatic activity
citrated, EDTA, and fluoride
a constant change in absorbance per unit time occurs only when the rate of the reaction is what type of order
zero order
in non kinetic assay, absorbance is made at how many second intervals
10 second intervals for 100 seconds
