Enzymes Flashcards
What is a catalyst? And what are the two types?
Catalysts are molecules that speed up reaction without being used themselves
1- biological molecules- Proteins and enzymes
2- RNA
Enzymes may require molecules. What are these molecules called? What are the two types?
Cofactors/coenzymes
Can be :
1- organic- biological molecules
2- inorganic| metal ions
Apoproteins- proteins
Holoenzyme- protein + coenzyme
What are the 6 classifications of enzymes?
Old - oxidoreductase
Tiger- transferase
Head butted - hydrolases
Little- lyases
Iguana - isomerase
Lamely - ligases
Explain
Oxidoreductase - transfer e as H
Transferase- transfers functional groups
Hydrolases - adds functional group to Water -cleaves cov bonds with water
Lysases- adds double bo;, clease covalent bond w/o water
Isomerase- isomerize by group transfer
Ligases- forms C-o,c-n,c-h,c-s . Coupled with ATP cleavage
Why are enzymes necessary
Accelerate
Regulate
Very specific , no side rxn
Main role in majority of rxn.
Provides an environment for easy bond form and breaking
ES and EP are cov or non-cov. Why?
Non Covalent
Because these complexes are reversible
Transition state theory
The energy required for substrate and reactant to reach the transition state
Describe ES stage
- S to ES is called binding energy
- S is bit distorted to look like product
Explain how s is distorted without needing extra energy.
1- E to ES is a energy releasing process, binding energy which is due to the formation of favourable interactions are formed- H bonds, hydrophobic bonds, VDW forces.
2- some of this binding energy is used for S distortion
3- ES start working from the ground level.
Compare the energy graphs
1- no enzyme
2- enzyme complementary to substrates
3x enzyme complimentary to transition state.
What are other affects of ES complex?explain?
-Reduce entropy of S -
-Dissolution - breaking the h2o shell around substrate
-Alignment of the groups that react
-induced fit- enzymes alter slightly to fit the transition S state, Affinity of S + is higher then S.
-strain reduction - steric / electrostatic are accommodated.
How and why does enzyme bind to only a few molecules?
1- shape consistency - 🔐
2- electrostatic consistency - correct matching of ionic and hydrogen bonds
3-thermodynamic consistency - the ability of substrate to flex to fit enzyme and vv.
Enzyme specificity is of two types?
1- optical specificity - different enzymes needed for D and diff for L
2- geometric specificity - different enzymes needed for cis and diff for trans.
Draw the two examples of the pervious specificity
Explain why metal ions are important ?
- week interactions between metal ions and substrate stabilize charged transition state and orient and bind the substrate
- megals accept and donate e in redox reactions
Describe the mechanism of carboxypeptidase
Carboxypeptidase use zinc as an alternate to amino acid to form an oxyanion hole
Draw the carboxypeptidase equation
Name the 3 types of enzyme inhibition
1- competitive
2- uncompetitive
3- non competeive
Explain competitive inhibitors and name a few examples.
Attach to the active site of the enzymes
Lipitor, viagra, protein inhibitors, VCT, LVV PETER
Malonic acid is an inhibitor of succinate dehydrogenase
Describe the changes of Vmax and kmax and why they occur.
Vmax is unchanged
- the rate decreases as the concentration of substrate increases, However at very high substrate concentration, the ability / prob of an inhibitor to bind to an enzyme is = 0. Substrate displaces I
Kmax increases
- kmax is apparently increased because now it takes more [S] to reach the vmax
Draw the MM and LB plots for competitive
Describe uncompetitive inhibitors
I binds to the allostseric site of ES complex only
Eg roundup
Dexcribe and explain the changes of Vmax and Kmax
Vmax is lower - inhibitor dec the active Etotal
Kmax is lower - higher affinity for (s) , the [s] to reach 1/2 Vmax is lower
Explain Non competivie
I binds to ES and E complex and prevent P formations
Explain change in vmax and kmax
Vmax decreases
Bcz I decreases E tot (active Enzyme), Vmax =kcat(Etot)
Kmax unchanged
The affinity of I to E and ES is same
Draw the two graphs for the other two inhibitors
State the curve name of
1- v0 by (s) graph
2- m-m grap - v0 by (s)
3- draw them
1- sigmoidal
2- hyperbole
Why are the curve different?
Sigmoidal is over a very narrow period of time therefore the enzyme acts as an in and off switch
When does this Non M-M graph behaviour occurs (i think they are talking about sigmoidal graph curve)
1- due to cooperativity
2- the binding of S to an active site , make the binding of a subsequent S easier.
What is cooperatiivity?
Occurs in a multi subunit enzyme
When the binding of an active site of one subunit, affects the binding of active site of other subunit.
How does binding of one S to an active site affects the binding of the other S of the active site?
-in a multisubunit enzyme, Each subunit can occur in 2conformations
T-Etaut (low affinity) and R- Erelaxed (high affinity)
-when there are few S, The equilibrium favours T, low affinity
- when there is S and s binds, the equilibrium favors R phase - high affinity of S , and higher active sites so more S binds! Untill enzyme is saturated
-this causes the sigmoidal curve
Draw what was explained regarding the non behaviour of mm curve
What can affect the kinectic curves - the sigmoidal and hyperbole curve
Anything that affects the equilibrium
Eg - activators and inhibitors
How do activators and inhibitors affect kinetic curve?
I and A bind to allosteric sites
Inhibitors bind to the T form and shift equilibrium to T form , bcz less S binds
Activators and substrates bind to R form and pull eq towards R
Give an example of I and A binding and also draw the curve
Enzyme- phosphofructokinase
Substrate- fructose 6 p
Inhibitor - ATP
activators- AMP
Give an example of allosteric enzyme /non enzyme and how it helps the human body
Allostericity helps extracts o2 from mothers blood
Hemoglobin isnt an enzyme however it binds to O2 with cooperatively, which affects how it responds to changes in o2 demands.
BGP and 2,3 DGP are inhibitors
Fetal hemoglobin has low affinity for BPH and higher affinity for o2 then adult haemoglobin
Therefore it has to absorb o2 from mothers blood.
Explain the catalysed and uncatalysed reaction of carbonic anhydrase
Uncatalysed
The ocean water has co2 , which decreases its PH , makes it more acidic
H2O + CO2 —-> HCO3- + H+
Catalysed by carbonic anhydrase
H2O-E-CO2 <—-> E-HCO3- + H <—-> E +HCO3-
10^6 time s more faster
Does a catalyst affect keq, why or why not?
The ratio of product conc to reactant conc
No
If the forward rxn increases, the backward rxn increases too because the free energy from the product to the transition state will also be lower
Why is studying enzyme mechanism important?
Provides knowledge and understanding basis of life, for modern drug dev, medicine and agriculture
Give an example of disease and drug that was made using protease studies/enzymes,proteins studies.
HIV RNA GENOME — > HIV polypeptide——HIV PROTEINASE—-> breaks them down , which become active and lead to virus formation
Structure based drug design and enzymology was used to made VIRACEPT, HIV proteinase inhibitors.
What are the two ways rates are measured experimentally
Increase in [P] over time
Decrease in [S] over time
What happens if we
1- lower the Ph from optimum
2-lower the temp from optimum
3- increase ph from optimum
4- increase Temp from optimum
1- enz inactive
2- enz inactive
3- enzyme denatured by ionic disruption
4- enzyme denatured by heat disruption
What type of curve is a M-M curve ? Why does this curve occur?
Hyperbole
Rate is dependent on two vectors
1- the time taken for substrate molecule to reach the enzyme- varies with the conc of substrate
2- the time taken for the enzyme to process substrate to the product - fixed
-Rate inc (V) as [S] increases
However as the [S] increases a lot , the amout of time taken for the substrate to reach the enz is 0
And the rate is now dependent on the processing time only which is constant
At this stage we see a constant Vmax with inc substrate conc and enzyme is known to be saturated
This causes a hyperbole curve
What are the assumptions made during the derivation of the mentis equation?
1- the k3»>K4, so k4 isnt a factor (this situations occur earlier in the reaction, when [P] =0)
2- K1 > k3, this make E+P formation the RDS( k3 - rate determining step)
3- rate of formations of E.S = k1[E][S]
4- rate of breakdown of E.S= k2[E.S] + k3[E.S]
5- in an equilibrium- the rate of forward formation=rate of backward breakdown
And equalize the above two equations.
What are the 3 main reasons , that Km is important?
1- shows affinity of E for S
High km - fast dissociate- low Affinity -need for high [S]
Low km- slow dissociate- high affinity- need for low [S]
2- kx = [S] at 1/2 Vmax
High km , a need for high [S]= unfavourable reaction
Low km, need for low[S] - favoured reaction
3- km=[S] , [E.S]=[E]
Why cant you ever calculate the Vmax from the M-M curve?
Vmax is a horizontal assimptote
The curve will come closer to Vmax , but never touch Vmax
How can you accurately calculate Vmax from a graph?
Line weaver burke plot graph and its line equation
Bcz -, although Vmax is never reached , it can be extrapolated to straight line.
- very sensitive to variation
Recall back , we mentioned the rate limiting step to be k3 (very slow). Why is rate limiting step important?
Helps us understand how many substrate convert to product in one second (turnover rate)
Quality control of vaccines
Help regulate optimum conditions in human bldy
Explain and derive kcat = Vmax/[E.tot] using kcat=k3
Kcat ranges from
5x10-1 —- 4x10^7/s
Explain the use of kcat/km❓❓
It is the specificity constant - shows the efficiency of the enzyme
Higher sc higher efficiency
When km»_space;>[S]
Then we can
V=kcat(etotal)(s)/km
What are the advantages pf allosteric enzymes?
-REGULATION IN TWO WAYS
1- regulates enzymes using end product inhibition or feedback inhibitions
-Helps in regulation, where the final product inhibits Enz 1
For ex L-Ile inhibits threonine dehydratase
-it acts as inhibitors, by not resembling as the substrate for Enz1, so doesn’t attach to active site
2- tight regulation, because of high sensitivity of[S] over a narrow timeframe
Thats how- Remeber how we get the sigmoidal curve in it inital time , instead lf a linear straight line impn initial time in M-M curve
What are other types of regulation ?
1-Covalent regulations
2-Zymogens
3-irreversible inhibitors
What are the 4 types of covalent regulations?
1-PROTEIN KINASE - (adds P to amino acids)
phosphorylates ser
Phosphorylated ser attaches to allosteric site (causing allosteric conformation change) of glycogen phosphorylase
2- this above can be reversed by removing P from ser by PHOSPHATASE
1-2- phosphorykatio/deohophorylation - activates enzymes in metabolic control mechanism
3- protein ubiquitination- addition/removal of ubiquinin to influence its activity
Eg- analogous to protein phosphorylation
What are zymogens
Zymogens are enzymes that are activated by proteolytic cleavages
What zymogens does pancreas produces
Chymotripsinogen, tripsinogen, proelastase , procarboxypeptidase
What enzyme carries out proteolytic cleavage of the zymogenes produced in pacreas
Enteropeptidase cleaves AA 1-6 of trypsinogen
Tripsinigen then activates other proenzymes that help in digestion of proteins on the duodenum
What are irreversible inhibitor?
Irreversible I form cov bonds with the active site AA
Give 3 examples of irreversible inhibition
1- penicillin reacts to serine in trans peptidase , inhibiting the bacterial cell wall synthesis
2- asperin acetylates ser in cox 2 enzyme ( that produces prostaglandins , a hormone that stimulates inflammation)
3- diisopropyl attaches ser 195, inactivating it
