Enzyme properties, kinetics and regulation Flashcards
Definition of an enzyme
Biological catalyst, increase R of R without altering final equilibrium between reactants and products, v efficient
Definition of cofactors
Inorganic elements, often transition metals as oxidation state can change
Definition of coenzymes
Not enzymes or inorganic ions but are still vital to enzyme function
Definition of isoenzymes
Different protein structures but catalyst same reaction
Definition of enzyme kinetics
The study of the rate of an enzyme catalyses reaction and how that rate varies with different substrate concs, amount of inhibitors, metal ions and cofactors, pH
Definition Kcat
Turnover number is equivalent to no of substrate molecules => product in a given unit of time on a single enzyme molecule when enzyme is saturated (s-1)
Definition of competitive inhibitors
Block enzyme AS, does not actually bind
Definition of non competitive inhibitors
Interfere in some other way, can be reversible or irreversible
Doesn’t actually change shape of AS, changes bonds within AS
Process of enzyme catalysis
S complementary to AS Greater affinity between S and AS to form ESC Catalysis Lower affinity between S and AS in EPC Product released from AS
Consequences of enzyme specificity in a compartment
Results in complex coordinated metabolic pathways
Classification of the enzymes based on catalytic reaction
Further divided into groups according to substrate/source
With 4 digits
Classification on enzymes
Oxidoreductase, Transferase, Hydrolase, Lyase, Isomerase, Ligase,
Oxidoreductase
Oxidation or reduction
Transferase
Transfer functional groups from donor to acceptor
Hydrolase
Hydrolysis w H2O
Lyase
Groups to C=C, cleavage of C-C, C-O, C-N
Isomerase
Isomerisations in same molecule
Ligase
Form C-C/C-N w ATP cleavage
Naming of enzymes
Substrate reaction type + are
No at front=class
Enzyme structure
Proteins, 1+ polypeptide chains w 3D structures
Stabilized by weak HB, ionic bonds, hydrophobic interactions, easy to break
Sensitive to environmental changes, can denature
AS has functional groups that stabilize transition state of reaction
Bonds maintain 3D structures of AS
Lock and key model assumptions
Assumes enzyme and AS is rigid
Modifies lock and key model assumptions
AS does not have to be completely complementary to S
As S moves into AS, S distorted so it is complementary
Increase in energy level of S, decrease in free energy of product
Induced fit model
Shape of AS and S changes, less competition from other molecules
As will only bind to specific functional groups
Catalytic triad in chymotrypsin by hydrolysis
Enzyme creates nucleophile from serine side chain
Nucleophile attacks substrate
Covalent intermediate is formed with second product bonded to serine and first product released
Enzyme creates a nucleophile from water molecule
Nucleophile attacks covalent intermediate, breaking covalent bond to serine
Second product is released
Effects of temperature on enzyme reactions
Increased temp, increased KE, increased collision theory
Increased optimum, AS denatures, no further reactions can take place
Effect of pH on enzymes reactions
Optimum pH can cary depending on the physiological compartment it works in
Common enzyme cofactors
Cu2+ Fe2+/3+ K+ Mg2+ Ni2+ Se Zn2+
Enzyme coenzymes
NaD+ => NADH + H+
FAD => FADH2
ATP => ADP + Pi
What makes isoenzymes different to normal enzymes
Different genetic code, catalyses same reaction with different protein structures
Often found in different cellular compartments or different amounts in different tissues
Have distinct biochemical roles
Rate of reaction equation
change in product/change in time = rate (v)
Rate of reaction to conc of substrate
Starts linear, rate proportional to substrate conc
Increases substrate conc, rate plateaus out
Ends asymptotically, will never work at max rate
Michaelis Menton reaction model
k1 k2
E + S <=> ES => E + P
k-1
Michaelis Menton reaction model assumptions
[S] > [E] so amount of substrate bound to enzyme at 1 time is small
[ES] does not change with time
Initial rates used
E + S <=> ES
Michaelis Menton equation
Vo = Vmax [S] / Km + [S]
Vmax = max rate when all AS saturated Km = k-1 +k2 / k1
Km values and its significance
Higher Km = lower affinity between E and S
Michaelis Menton plot
When Vmax/2 = Km = [S]
Kcat, turnover number
Equivalent to no of substrate molecules converted to product in given unit of time on a single saturated enzymes (s-1)
How to compare catalytic efficiency
Neither Km, Kcat are alone sufficient to compare catalytic efficiency of 2 enzymes
Kcat/Km, specificity constant, provides best method
What is the ideal value of Kcat/Km?
Be as high as possible!
Lineweaver Burke plot equation
MM plot/1
1/Vo = Km/Vmax [S] + 1/Vmax
What does the x intercept mean on LBP
-1/Km
What does the y intercept mean on LBP
1/Vmax
Clinical uses of enzyme measurements
Differential diagnoses of disease (investigating plasma levels of escape enzymes)
Lab estimations of metabolites (glucose in body fluids)
Describe the 5 forms of lactate dehydrogenase isoemzymes
4H 3H1M 2H2M 1H3M 4M
Why do you have different forms of the isoenzymes
Need different monomers for operating in different compartments
Low Kcat, low Km or vice versa, depends on conditions
Use of isoenzymes
Electrophoresis of LDH
Different tissues have different levels of each LDH, diagnostic tool
Competitive inhibitors, what changes in MM plots
Km altered as affinity of S changes
As [I] increases, affinity decrease
As [s] increases, affinity increases
Vmax/2 does not change
Non competitive inhibitors, what changes in MM plots
Vmax altered as maximum rate cannot be reached
Km does not change
No effect on Km as AS not affected
Vmax/2 decreases with inhibition
Chemical uses of enzyme inhibitor
ACE is an enzyme that converts angiotensin I => II
Captopril inhibits ACE, treats heart failure, causes vasodilation and BP falls
Increased acetylcholine, increase cognitive impairment Acetylcholine esterase (reversible/irreversible) can breakdown ACh
How to regulate enzyme activity
Allosteric binding sites
Covalent modification by other enzymes
Induction, supression of enzyme synthesis
Allosteric regulation and binding
Results in sigmoid curve due to cooperative binding
Can be positive, increase chance that S binds to AS
Can be negative, decrease chance that S binds to AS
Covalent modification by phosphorylases/kinases
Can both activate and deactivate enzymes
Can also result in catalysis of a different reaction
Can also involved other molecules (adenine, methyl, acetyl)
Regulating enzyme activity
High levels of substrate => high levels of substrate that increase rate of synthesis of key enzymes
When substrate availability changes, what changes and time frame
Rate
Immediate
When product inhibition occurs, what changes and time frame
Vm, Km
Immediate
When allosteric control occurs, what changes and time frame
Vm, Km
Immediate
When covalent modification occurs, what changes and time frame
Vm, Km
Immediate to minutes
When synthesis/degredation of an enzyme occurs, what changes and time frame
Amount of enzyme
Hours to days
What cofactor does carbonic anhydride need
Zn2+
What cofactor does glutathione peroxidase need
Se
What cofactor does urease need
Ni2+
What cofactors does pyruvate kinase need
Mg2+
K+
What cofactors does cytochrome oxidase need
Fe2+/Fe3+
Cu2+
