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Allosteric Regulation and enzyme kinetics
Terms in this set (20)
Oxygen binding vs loss of electrons of Hemoglobin
- Oxygenation: reversible binding between Iron ion and O2 in lungs for other tissues
-After forming Fe2+O2, Iron donates an electron to become more positive and Oxygen O2-. O2- becomes metHb (occurs when red colored blood irreversibly becomes brown)
Structure of hemoglobin
-Four units (four myoglobins), symmetry of 2-fold axis of rotation- central cavity, oxyHb and deoxyHb are not superimposable
-each unit has a heme group is a protoporphyrin IX structure. Hexavalent iron in the middle of heme. Iron is connected with four bonds from Nitrogens. One bond comes from the protein (proximal Histidine). The sixth position is where oxygen binds. Distal histidine is not bound to iron and is on the other side.
-Adding more oxygen makes it easier for oxygen to bind and form oxyhemaglobin. S binding curve. (positive cooperative binding)
-Increase in oxygen content from artery to vein. S- binding curve depicted the allowance of more oxygen to be delivered to arteries
1/Kd= Y or fractional saturation
p50= half of the molecules are saturated
Hill plot is adjusted for multisubunit binding which myoglobin slope is one and hemoglobin is a positive sigmoidal. Hill equation assumes "all or nothing" binding.
For hemoglobin, the slope of lower asymptote is p50 of first oxygen. The slope of upper asymptote is p50 of last oxygen bound
For both hemoglobin and myoglobin, the slope at 1 is the Hill coefficient to compare effects of allosteric effectors.
Adair- takes hybrids into account
Structure of myoglobin
-A heme protein, single polypeptide chain. No change in structure of oxy or deoxy myoglobin. single curve
-No real change in oxygen binding from artery to vein
The Perutz Mechanism
T- state- tense without oxygen- histidine pulls iron out of the plane
R-state- relaxed with oxygen- oxygen pulls iron into the plane
-Interactions between like subunits is limited
-Interactions between unlike subunits involve hydrophobic and ionic bonds
-oxygenation induces a rotation at the a1B2/ a2B1 interface
-Salt links anchored protein. Some salt links break going into R state. Oxygen binding to iron provides enough energy to break salt links to help hemoglobin transition from T to R state. The addition of the 1st oxygen is less energetically favorable than the 2nd oxygen because there are more salt links to be broken. Reversing this thought-> positive cooperativity
What is Allosterism?
An effector or modulator influences the binding of ligand to the ligand binding site by interacting with another site (allosteric site) on the protein
Homotropic vs heterotropic effect
If the 2 ligands are identical (binding to active site)- homotropic effect- O2 and its positive cooperativity where oxygen as the effector and ligand interacts with another site to also bind oxygen as a ligand
If 2 ligands are different- heterotropic effect 2,3-BPG. It's binding allows oxygen to bind the next subunit through positive cooperativity
What is positive/ negative cooperativity?
Positive cooperation- effector increases ligand affinity and binding
Negative cooperation- effector decreases ligand affinity and binding
Hemoglobin purified vs in whole blood
- both have S shape of pressure of O2 in hemoglobin and saturation of O2 is hemoglobin. Purified hemoglobin left shifts.
-In whole blood, 2-3 BPG binds T state in place of oxygen to help create R state (broken salt bridges. When R state is formed, oxygen can easily bind. Shift equilibrium to R state. 2-3 BPG is a heterotropic negative allosteric mediator of O2 binding to Hb causing positive cooperativity
What shifts hemoglobin saturation of oxygen to more oxygen binding and disocciation?
Lower pH, Higher CO2
Models for Allosteric Protein function
Symmetric Model- allosteric proteins are composed of subunits resulting in a symmetrical molecule. Each subunit has more than one conformation. The ligand can bind both states with different affinity. Molecular symmetry is conserved during conformational changes (hetero and homotropic effect) symmetry does not allow hybrids or negative homotropic effects
Sequential or induced fit model- ligand binding induces conformational change in subunit which in turn influences the conformation of the neighboring subunit (supports + and - cooperative effects). No equilibrium between conformation states allowing hybrids.
k= the rate constant- characterizes reactivity
Rate of reaction- velocity- rate at which product is formed or lost
rate of the reaction is proportional to the concentration of reactants (like lechatlier)
A to B is first order with respect to A. Log of first order is a straight line.
2A to B is second order with respect to A. Inverse of initial concentration is linear.
A+B to C is first order with respect to A or B and 2nd order overall
Concerning Kinetic energy, when do molecules typically react and how do enzymes help
Way past the max level of entering molecules, once kinetic energy is high enough
Enzymes decreases the energy required for the reaction to occur. It doesn't change the number of molecules.
Enzyme kinetics- Michaelis Menten Equation
Initial rate/ velocity increases as substrate concentration increases
Plotting initial velocity against substrate concentration is a positive linear relationship
With enzymes, you don't need to keep adding substrate, it levels out against reaction velocity like a zero order
Michaelis-Menten form equation with enzyme intermediate
Michaelis- Menten- Briggs- Haldane equation- Express [ES] in terms of kinetic constants, initial enzyme and initial substrate concentrations
What are the Assumptions and Restrictions of M-M-B-H
1) Consider only initial rates
2) Substrate concentrations is greater than enzyme concentrations. Initial substrate= free substrate
3) ES to E +P is rate limiting step so initial velocity= k3 [ES]
4) Enzyme is conserved
5) [ES] is in a steady state
How to characterize the Michaelis-Menten graphs
Km=[S] at which v= vmax/2 and decribes affinity of S for E
Km=Michaelis constant and is obtained assuming concentration of substrates is greater than Km
How do you linearize the Michaelis-Menten graph
Inverse of initial velocity
Inhibition of Enzyme activity
Decreasing vmax and increasing Km (concentration) inhibit
competitive- changes Km
uncompetitive- vmax and Km
Mixed-vmax and Km
Enzyme reaction mechanisms Cleland terminology
ABC- Substrates in order they bind E
PQR- Order they leave E
EFG stable forms of the enzymes
Number of products- uni, bi, ter
What to know
definition of allosteric mediators
Allosteric sequence sequential
first- second order reactions
how they change in substrate concentration
Efficiency at which enzyme works
Mechanisms and interactions with inhibitors
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