Module 6 Flashcards
overview of enzyme
What does the configuration of enzyme do
Provide a specific environment i.e. active site for a given reaction to proceed rapidly
What are the active site of enzyme
They are lined with functional groups that binds to substrate
What is the role of enzyme
Lower the activation energy required and perform this function with high degree of specificity
How does enzyme catalyze reaction
By forming a complex between enzyme and substrate (ES)
What is a catalyzed reaction coordinate
E + S <> ES <> EP <> E + P
What are the cataliytic mechanisms
–acid-base catalysis:give and take protons
–covalent catalysis:change reaction paths
–metal ion catalysis:use redox cofactors, pKa shifters
Why are enzyme important?
-convert carbon fuel source into cellular energy in a appropriate timescale
-Forming ethanol from pyruvate.
Pyruvate— pyruvate decarboxylase–> acetaldehyde — alcohol dehydrogenase–> ethanol
What disease can be caused regarding enzyme?
Excessive or deficiency in enzymatic activity.
e.g. Phenylketonuria is caused by a deficiency in the enzyme Phenylalanine Hydroxylase
How does most drug work?
They target enzyme by inhibiting or activating the target
Does enzyme work alone
not all
What can enzyme work with?
cofactors (e.g. MG2+, K+)
coenzymes (carries functional groups) e.g. Coenzyme A, FAD, NAD, biocytin
What type of enzymes are there?
8 types – Kinases – Phosphorylases – Phosphatases – Dehydrogenases – Mutases – Isomerases – Hydratases – Synthases
What is kinase (K) for
Catalyses the phosphoryl transfer from one molecule (usually ATP) toanother;e.g.Hexokinase
What is phosphorylase (P’Lase) for
Catalyses the covalent addition of inorganic phosphate(Pi)to a molecule; e.g.Glycogen Phosphorylase
What is Phosphatase (P’Tase) for?
Catalyses the cleavage of a phosphate to yield the dephosphorylated product and Pi;e.g.Glucose-6-phosphatase
What is Dehydrogenase (DH) for?
Catalyses an oxidation/reduction reaction commonly using NADH/NAD+, NADPH/NADP+ or FADH2/FAD as cofactors; e.g.Glyceraldehyde-3-phosphate
dehydrogenase
What is Mutase (M) for?
Catalyses the shift of a phosphoryl group from one atom to another within the same molecule;e.g.Phosphoglycerate mutase (tetrameric in yeast and dimeric in bacteria)
What is isoemrase (I) for?
Catalyses the conversion of one isomer to another; e.g. Triose Phosphate Isomerase
(step 5 of glycolysis)
What is hydratase for?
Catalyzes the addition/removal of water
e.g. enolase
catalyses the conversion of 2-phosphoglycerate to phosphoenolpyruvate
What is synthase for?
Catalyses the synthesis of a product. e.g.citrate synthase.
Addition of an acetyl group from Acetyl-CoA to oxaloacetate to form citrate in the first step of the Krebs (citric acid, TCA) cycle
What prevention does enzymes have
energy barriers to prevent complex molecules from reverting spontaneously, reduce activation energy and thereby increase the rate of reaction
Characteristic of Enzyme
Do not affect : delta G
transition states are transient species
What is binding free energy?
the difference between the activation energies of the uncatalysed and catalysed reactions caused by the enzyme binding the transition state
How do enzyme reduce the activation energy and accelerate rates of reaction
– binding substrates in the correct orientation relative tot he active groups
-providing catalytically active groups(side chains, acids, bases, metal ions)
– Polarizing bonds, stabilising charged species (usually unstable)
– stabilizing the transition state
Weak binding interactions between the enzyme and the substrate provide a substantial driving force for enzymatic catalysis
The rate decreases with time due to a number of possible reasons(or a combination)
– (main reason) S is depleted by conversion to product
– The reaction is reversible, so as [P]↑the rate of the reverse reaction↑
– The enzyme may be unstable under the reaction conditions
What is the beer-lambert law
∆A = εx ∆c x l
What is the shape of the Michaelis-Menten plot
‘rectangular hyperbola’
What can is related to the rate of an enzyme catalysed reaction
proportional to enzyme concentration
What are the unit of the Michaelis-Menten plot
Initial velocity, Vo against subtrate concentration, [S0]
What is the michealis constant
When [S] is at ½Vmax, defined at Km, michealis constant
What is similar to km and [S] in enzyme kinetics
Kd and [L] in binding of protein and ligand e.g. Hb and O2
What is the michaelis-menten equation
Vo= (Vmax[S])/(Km+[S]
What is the Rate of formation of product Vo
V0 = k2[ES]
What is the Rate of ES formation
Rate of ES formation = k1[E].[S] = k1([Et] –[ES]) [S]
What is the chemical equation of Michaelis Menten Theory
E + S k1> E + P
What is the Rate of ES breakdown
k-1[ES] + k2[ES]
What assumption do we use for the Michaelis Menten Theory
Assumption : ES conversion to E +P irreversible
Assumption : Steady-State Conditions [ES] constant
Assumption : [S]»_space; [Et]
Assumption : [S]»_space; [P] (initial conditions)
What is the rate determining step
V0 =𝑘2 [𝐸𝑆] Vmax
What is the equation for maximum velocity Vmax
Vmax = k2[Et]
What is the steady-state assumption
Vo represents a ‘steady state’ where [ES] remain constant
The rate of formation ES = the rate of ES breakdown
What is the ratios of concentrations OR ratios of rate constants
[E][S]/[ES] = (k2 + k-1)/k1, defined as Km, steady state concentration, not equilibrium concentration
What is a double reciprocal plot
it is the lineweaver-burk analysis
How is Km related to Kd
They have the same form for them chemical equation
What kcat or turnover number mean
k cat)is defined as:• the number of molecules of substrate converted to product (S to P) per unit time per enzyme molecule saturated with substrate(i.e.when[ES]=[Et] k2, usually the rate limiting step
What the specificity constant is and what does it mean
the rate constant for conversion of E + S to E + P (kcat/Km)
What is the formation of ES at equilibrium Ka
1/Kd
What is Kd
Kd = (k-1)/k1 = [E][S]/[ES] , how tight the enzyme tie to a substrate
What is Km
The overall reaction for ES, an indication of the affinity of the enzyme for S. at steady state condition and take account of the catalytic step
What is low Km
high affinity of the enzyme for its substrate
Which Km of substrate have 30 fold difference in affinity for hexokinase
d-glucose = 0.05 mM, d-Fructose = 1.5 mM (in glycolysis)
What does the Km usually related to
its own concentration in the environment
When [S] «_space;Km
Vo is proportional to kcat/Km
What does kcat/Km reflect
both substrate affinity and catalytic efficiency (higher indicates more efficient use of the substrate
Describe the difference between irreversible and reversible inhibitors
bind covalently to the active site, destroy a functional group essential for enzyme activity, or form a stable noncovalent complex with the enzyme.‘Suicide inhibitors’
Bind reversibly to enzymes and inhibit the enzyme either by competitive, uncompetitive or mixed modes of inhibition.
What is the apparent Vmax and Km of competitive inhibition
Vmax, alphaKm
What is the apparent Vmax and Km of uncompetitive inhibition
Vmax/alpha’, Km/alpha’
What is the apparent Vmax and Km of mixed inhibition
Vmax/alpha, alphaKm/Alpha’
Describe the mechanism of a competitive inhibitor
Inhibitor can bind to the same site, preventing substrate from binding. The activity is zero
List the properties of the factor α competitive inhibitor
It is the enzyme that was used by the inhibitor. The factor of enzyme dropping.
The more enzyme removed by inhibitor
Describe how α changes the apparent Km competitive inhibitor
When alpha is 1, no inhibitor.
As alpha increase, Vmax not changing, Km change, apparent Km increase.
Generate and analyse Michaelis-Menten and double reciprocal plots that show the effect of a competitive inhibitor
steeper as inhibitor increase. Showing as if the enzyme is binding more weakly to substrate, but in fact it binding to inhibitor
calculate α from kinetic data of competitive inhibitor
Vo = Vmax[S]/(alphaKm+[S])
What is uncompetitive inhibitor
Does not compete with the same binding site but recognise and binding to the enzyme-substrate complex. No activity. IES, constant is Ki’. Decrease effectiveness of the production of product. does not contribute
Describe how α’ changes the apparent Vmax of uncompetitive inhibitor
alpha become 1.5 or more, y axis increase. Vmax look smaller
Describe how α’ changes the apparent Km of uncompetitive inhibitor
Decreases, but actually it does not change
Be able to calculate α’ from kinetic data of uncompetitive inhibitor
Vo = Vmax[S]/(Km+alpha’[S])
Generate and analyseMichaelis-Menten and double reciprocal plots that show the effect of an uncompetitive inhibitor
increase in parallel lines, Km decrease, Y axis increase.
What is mixed inhibitor
It can bind to both the free-enzyme and the enzyme-substrate complex with constant of Ki and Ki’(ES complex) binding at different site. activity is zero. can bind to either side
List the properties of the factor α and α’ in mixed inhibitor
alpha is binding to free-enzyme, alpha’ is to enzyme-substrate complex
michaelis equation for mix inhibitor
Vo=Vmax[S]/(alphaKm+alpha’[S])
What is the apparent Km of mix inhibitor
decrease(depends of the relationship between alpha and alpha’
What is the apparent Vmax of mix inhibitor
decrease
Describe the features of an allosteric enzyme
Regulate metabolic pathways by changing activity in response to changes in concentration of molecules around them
Explain how positive and negative allosteric modulators control enzyme activity
Positive allosteric enyme bind to regulatory,change the conformation at the active site and help S bind with higher affinity
Describe how different allosteric modulators stabilize different conformational states
Binds to the regulatory site
Analyze plots of kinetic data for allosteric enzymes to identify the action of allosteric activators and inhibitors
High activity R state k0.5 decrease, Low activity T state K0.5 increase
Use the example of the ATCase from E. coli as an example of allosteric regulation
S is Asp, sigmoidal V0 vs S, large increase in Vo. First step to produce nucleotides UTP and CTP. When high level CTP inhibit ATCase, negative modulator. ATP is the positive modulator
What are Allosteric enzymes are regulated by
compounds called ‘allosteric modulators’ or ‘allosteric
effectors’
Can allosteric modulator bind reversibly to enzyme
Yes, and non-covalently
Is kinetic of allosteric regulators same as Michaelis-Menten kinetics?
No
How many subunit does ATCase have
complex quaternary structure of 12 subunits
6 catalytic subunits, arranged as 2 x trimeric complexes. The catalytic subunits function cooperatively
And – 6 regulatory subunits, arranged as 3x dimeric complexes
Integrate concepts of primary, secondary, tertiary and quaternary structure to describe the structure of α-chymotrypsin
5 disulfide bonds, hydrophobic pocket that binds the side chain of Phe,Tyr or Trpin the substrate protein. active site is described as a ‘catalytic triad’of Asp102, His57 and Ser195
Rationalize the optimal pH for α-chymotrypsin activity with the protonation/deprotonation of His and the N-ter of the B chain
Below pH 7, His is protonated and cannot accept proton from Ser, kcat decrease. Above pH 8, His is all deprotonated, kcat does not change
For maximum activity ,His57 must be unprotonated (i.e. >pH7) and N-ter of the B chain (Ile16) must be protonated (ie
What is Chymotrypsin
proteases that cuts peptides at specific locations on the peptide backbone, cleave the peptide bond adjacent to aromatic amino acids.
