Enzymology Flashcards
study of Enzymes
ENZYMOLOGY
catalyzed reactions are specific and essential to physiologic functions like
- hydration of CO2
- nerve conduction
- nutrient degradation
- energy use
Enzymes that facilitate **coagulation **are specific to
PLASMA
Biological catalyst (enzyme) mostly inhibit
Homogenous catalysis
Catalytic site; water free cavity
Active site
Allosteric site may bind
regulator molecules
How do these molecules inhibit or enhance substrate binding?
conformational change is introduced in the active site
Reaction rate is reduced when
there is conformational change due to allosteric inhibitor, unable to bind to enzyme and prevents it from lowering the activation energy
Cofactor and coenzyme bind temporarily and permanently through?
T: ionic and hydrogen bond
P: strong covalent bond
alters the spatial configuration of an enzyme for proper substrate binding
COFACTOR OR COENZYME
____ IS A TYPE OF COFACTOR
COENZYME
____ are covalently bound and can be removed by ________ the enzyme
some cofactors, denaturing
thermostable and form the active site of enzyme
Cofactor
true or false: coenzyme can catalyze a reaction by itself
false; coenzyme cannot catalyze by itself but has to bind to apoenzyme
Holoenzyme
coenzyme + apoenzyme;
comple and active form of enzyme
protein part of the enzyme; inactive form
apoenzyme
Catalytic components of DNA polymerase enzyme
apoenzyme
multi-subunit complex of DNA polymerase
holoenzyme
true or false: catalytic activity of enzymes depends on the **integrity of the enzymes’ 3D structure **
true
factors that act as denaturing agents
- temperature
- pH
- chemicals
denaturation disrupts the ___ bonds
hydrogen bonds (that stabilize the 3D structure)
Enzymes like polymerases resume their activity at temperatures higher than
90 degrees Celsius
causes the unfolding of the enzyme
extremes of pH
In extremes of pH, unfolding can lead to loss of structural integrity then
loss of function
____ bonding: secondary structure
____ bonding: tertiary structure
hydrogen; covalent
EC-IUB
Enzyme Commission of the International Union of Biochemistry
EC-IUB adopted a classification in ___, and was revised in ____ & ____
1961, 1972, 1978
EC CLASS 4
Lyases
EC Class 2
Transferases
reduction
gain of electrons
EC class of Lactate Dehydrogenase
Oxidoreductase
Class 1
conversion of ethanol to acetaldehyde in liver
Alcohol dehydrogenase
EC of Creatine Kinase
Transferase
In CK, a phosphate group from ATP is transfered to the _ atom of creatine phosphate
nitrogen
End products of CK
ADP and creatine phosphate
Cleavage of bonds with the addition of H2O
Hydrolases
EC of esterases
ACP, ALP
Hydrolases
group elimination to form double bonds (product)
LYASES
In Aldolases, it cleaves the 6-carbon molecule (fructose-1,6-diphosphate) to produce 2 3-carbon compounds:
glyceraldehyde-3-phosphate and dihydroxyacetone phosphate
True or false: Isomeric reactions are reversible
true
responsible for the conversion of carbohydrate, glucose, to toher compounds
Glycolytic pathway
EC class of mutases
Isomerase
triacylglycerol acylhydrolase
lipase
1,4-a-D-glucan glucanohydrolase
Amylase
In activation energy, reactants possessing enough energy to overcome the enrgy barrier participate in
product formation
Enzyme ___ free energy required to activate the reaction
reduces
true of false: transition state of the ES complex has a lower activation energy than transition state of the substrate aloe
true
Who are the two people who hypothesized the role ofsubstrate cocnentration in the formation of ES complex in 1913?
Michaelis and Mentern
Substrate readily binds to free enzymes at ____ concentration
low
FIRST ORDER: reaction rate is directly proportional to the ___________ concentration
substrate
further increase in substrate concentration no longer accelerates reaction due to
saturated enzyme with the susbtrate (no enzyme left to react)
Maximum velocity is achieved when
substrate concentration is high enough to saturate all available enzymes
Km
specifically the substrate concentration (enzyme yields 1/2 of the possible Vmax)
amount of susbtrate needed for a particular enzyme reaction
double reciprocal plot (yields a straight line)
Lineweaver-Burk plot
higher the enzyme level
faster the reaction
pH is carefully controlled at an optimal pH by
buffer solutions
low temperatures render enzymes
reversibly inactive
Common Activators (Inorganic Cofactors)
Metallic: Ca2+, Fe2+, Mg2+, Mn2+, Zn2+, K+
Non-metallic: Br-, Cl-
Common Coenzymes (Organic factors)
Nucleotide phosphates, Vitamins
true or false: Km is constant for each enzyme and can be altered
false: cannot be altered
presence of compettive inhibitors will ___ the maximum velocity
lower
bind to enzyme at a place other than the active site; metallic
noncompetitive inhibitors
true or false: both inhibitor and susbtrate bind to enzyme simultaneously
true
binds to the Enzyme-Substrate (ES) complex
Uncompetitive Inhibitors
example of antienzyme
Trypsin inhibitors
In rate of reaction, velocity constant o the reaction of the inhibitor with enzyme is
measure of the effectiveness of the inhibitor
convenient method of enzyme quantitation
measurement of enzyme activity (not directly the enzyme)
amount f susbtrate exceeds that of enzyme, rate of reaction will depend on the
ENZYME CONCENTRATION
Period for Analysis sequence
- Enzyme is initially introduced
- Excess substrate steadily combine with available enzyme
- Reaction rate increases
- Enzyme is saturated
- Rate of product formation, release of enzyme, and recombination proceed linearly
In fixed time, reaction is stopped, by inactivating enzyme with
weak acid
also known as Kinetic Assays
Continuous-Monitoring
Causes of deviation from linearity
- enzyme is elevated: substrate is used up too early
- coenzyme conc. is low
variables that may alter results
pH, temperature, substrate
Enzyme conc is usually expressed as
Units per Liter (IU/L)
alternativeof measurement of enzyme activity
measurement of enzyme mass
example of digestive enzyme where inactive precursors and inhibtors of catalysis are present in plasma
trypsin
enzyme activties in serum are due to
mixtures of immunologically distinct isoenzymes
enzymes can be used as
reagents
used for uric acid determination
uricase (urate oxidase)
In glucose determination, this enzyme reaction converts sugar other than glucose to their 6 phosphate esters
Hexokinase reaction
In glucose determination, this is used to monitor the change in reaction (catalyzed by G6PD)
Indicator reaction
equilibrium methods are also called
End point methods
Enzymes with high affinities for their substrate
- have low Km values
- most suitable for equilibrium analysis
general property of first order reaction
change in substrate concentration over a fixed time interval is directly proportional to its ratio of concentration
most accurate for enzymatic determination of substrates; more demanding than equilibrium methods
two-point kinetic methods
example of enzyme labels
ALP, Horeradish, peroxidase, G6PD, B-galactosidase
ENZYME-LABELED ANTIBODIES OR ANTIGENS
- react with ligand
- enzyme substrate is added
- washing then 2nd enzyme-labeled antibody is added
- Ag-Ab-enzyme complex is formed
- 2nd washing then substrate is added
conversion of susbtrate is proportional to
quantity of antigen
example of adsorbents
where immobilized enzymes are chemically bonded to
Microcrystalline cellulose, Diethylaminothyl (DEAE) cellulose, Carboxymethyl cellulose, Agarose
enzyme electrodes
enzymes incorporated into membranes
Ion-sensitive electrodes
when immersed in a solution of appropriate substrate, action of enzymes
produces ions to which electrode is sensitive
