Study Guide 6

What is the function of Carbonic Anhydrase? How does Hb help drive this reaction to completion?

Carbonic anhydrase will catalyze the reaction of H2O + CO2 → H2CO3. When reversed, the H2CO3 is deprotonated. Hb takes the H+ ions to protonate the histidines in the Bohr salt bridge converting Hb to its T-state which releases oxygen. When the protons are removed by Hb, the pH increases and shifts to produce bicarbonate. The removal of the proton will drive the reaction to completion because of Le Chatelier's principle.

Why is the effect of 2,3-BPG similar to the Bohr effect in deep tissues and in lungs? What does 2,3-BPG do to Hb?

2,3-BPG is an allosteric effector that will bind to Hb and stabilize the T-state due to the salt bridges in order to decrease the Hb affinity for oxygen and lowers pH. This works like the Bohr effect by creating small salt bridges to release oxygen. When the pH is lowered, salt bridges are formed and the protonation will cause the decrease in Hb oxygen affinity, releasing the oxygen.

To switch Hb from T to R, how many molecules of O2 must bind? What is the rule regarding O2 binding to Hb subunits and the T-R state switch?

A minimum of two oxygens must bind to Hb to favor the R-state. The quaternary structure of Hb favors the R-state only after oxygen has bound to one subunit of each alpha-beta dimer. The T-state is favored only after oxygen is released from one complete alpha-beta subunit.

What is a transition state analog? What type of inhibition would you expect from a transition state analog?

A transition state analog mimics and stabilizes the transition state to a point where it is more stable than the reactants and products. This allows it to be a competitive inhibitor to the actual transition state.

What is an acyl-intermediate (think covalent catalysis)?

An acyl-intermediate is a covalent enzyme-substrate complex.

What is an allosteric enzyme? What is an allosteric effector?

An allosteric enzyme can change their conformation ensemble when binding to an effector. An allosteric effector is a molecule that binds to a site other than the functional site on a protein and modulates activity.

What is an antibody? How do you make a catalytic antibody? How do catalytic antibodies increase the rate of a reaction?

An antibody is a protein that binds to a specific pathogen and activates specific immune response. Catalytic antibodies are made when we want to produce antibodies that only bind to the transition state of a chemical reaction. They are made by raising antibodies against transition state antibody and then purifies antibodies. It then gives the antibody substrate that the catalytic antibodies bind to weakly. The catalytic antibodies then distort the substrate into a conformation resembling the transition state. Here, a water molecule has attacked the carbonyl carbon to form the transition state. The antibody catalyzed this attack by stabilizing the transition state. The catalytic antibody releases the products. It will also catalyze the reverse reaction.

Be able to identify and interpret the trend of the Hb O2 binding curves at different pHs. If given a curve containing HbF and HbA, be able to identify which is which.

As pH decreases, the Hb O2 binding curves get less steep.

How does endurance training at higher altitudes affect an athlete's performance?

At higher altitudes there is less oxygen getting to athlete's tissues causing the body to produce 2,3-BPG by binding to a cleft in Hb and causing more O2 to be released in the tissues decreasing the Hb affinity for oxygen. This release of oxygen allows the tissues to perform more cellular respiration allowing the athlete's performance to be enhanced.

How can H146 be protonated at pH of 7.4 if the pKa of the His amino acid is 6.0?

Because His is in close proximity to the negative H146-D94 salt bridge, the microscopic pKa of His is lowered and protonated; therefore, causing it to be favored at a lower pH.

What is the evidence that Hb and Mb are homologous proteins?

Both Myoglobin and Hemoglobin are oxygen carrying proteins. Hemoglobin is found in red blood cells, and myoglobin is found in muscles. The major structural difference between Mb and Hb is Mb is a monomer, while Hb is a tetramer.

Do enzymes make unfavorable reactions favorable? Do they affect Keq? What do enzymes do?

Enzymes can couple an unfavorable reaction with a favorable one. They do not affect Keq because they only increase forward and reverse reaction rates.

What amino acids are good proton donors or acceptors?

Good Donors: asparagine, glutamine, histidine, serine, threonine and tyrosine Good Acceptors: aspartic and glutamic acid

A displacement mechanism (SN2) inverts stereochemistry, a dissociative mechanism (SN1) results in a mixture (retains and inverts). How does HEWL use SN2 to retain stereochemistry?

HEWL performs two consecutive SN2 mechanisms which individually invert stereochemistry but together retain it.

Describe how the Bohr Effect functions to get oxygen to working muscle

Hb is pH sensitive. In the presence of a tissue that is working to release CO2, the environment will be acidic. The low pH will cause Hb to decrease its affinity for O2 since the Bohr salt bridge will be formed. The salt bridge then will stabilize the deoxyhemoglobin and decrease the affinity. Hb then releases O2 around the tissue, like working muscle.

Why does HbF have a different affinity for oxygen relative to HbA? Why is this important to a fetus?

HbF has a higher affinity for oxygen relative to HbA to facilitate the transfer of oxygen from maternal and fetal circulations in the placenta. This is important because the fetus's cells need oxygen to grow.

Name two ways that a catalyst (not necessarily a protein) can accelerate a reaction. Which of these two ways is affected by an entropy trap?

Increasing the collision frequency or lowering the activation energy. An entropy trap affects collision frequency because it reduces rotational and translational freedom of substraets to increase productive collisions.

What is meant by 'induced fit'?

Induced fit refers to enzymes that have evolved flexible active sites that have a higher affinity for the transition state.

What would happen to Oxygen delivery if the D of the Bohr Salt Bridge where mutated to an L?

L, Leucine, is not anionic like D, aspartate, so no salt bridge would form with a cationic histidine. Therefore, Hb would favor its R-state and oppose the release of bound oxygen as a result.

What are the proximal and distal histidines? How does Hb keep O2 from oxidizing the Fe+2 iron?

Proximal His is a highly conserved His residue that is directly attached to the iron. On the other hand, distal his interacts with oxygen in the opposite face and is present when heme is bound to myoglobin increasing the affinity for O2. A hydrophobic cleft is formed around the oxygen and prevents the oxygen from oxidizing the iron. The hydrophobic cleft makes the environment nonpolar. If the oxygen oxidizes the iron, it creates a superoxide ion, which is unfavorable in a nonpolar environment. If the superoxide would form an ionic bond with iron, it would be prevented from leaving unless the ionic bond were to be broken, which costs a lot of energy.

What is the proximity effect? What is meant by "entropy trap"? What is a fish ladder?

Proximity effect is the binding and proper orientation of reactants increases effective concentration of reactants increasing rates Entropy trap reduces rotational and translational freedom of substrates to increase productive collisions A fish ladder is where substrate binding occurs at a local minima

Know what the R and T states mean. What is the molecular switch that changes Hb from the T-state to the R-state? What is the purpose of the T-state of Hb in tissues?

R state refers to the relaxed state of Hb, and T state refers to the tense state. Oxygen is the molecular switch between states. The R state is favored in oxygen rich environments, while the T state is favored in oxygen-deficient environments. The T-state is significant in tissues because changing Hb to the T state from the R state facilitate the release of oxygen into tissues.

Know the four 'tricks' enzymes use to enhance reaction rates.

Substrate binding: entropy traps, fish ladder, ground state destabilization Stabilize transition state: distort, strain, polarize, stabilize Position catalytic residues or cofactors: acid/ base, catalysts, nucleophiles, redox Use of covalent intermediates: fish-ladders, modify substrates (make bad leaving groups good), use covalent catalysts

What is the Bohr Effect? Draw the Bohr saltbridge and F-helix at low and high pH (include the heme, proximal and distal Histidines)

The Bohr effect is a decrease in the amount of oxygen associated with hemoglobin and other respiratory compounds in response to a lowered blood pH from an increase in carbon dioxide in the blood

Know the difference between oxygen binding curves for Mb and Hb. What is the physiological significance of the sigmoidal curve for Hb?

The Mb curve suggests that there is one Kd. The Hb curve suggests that it is cooperative meaning that binding at one Hb site increases the affinity of an oxygen binding to another Hb site. The Hb curve is pH sensitive meaning that a lower pH will cause the binding affinity and dissociation to decrease. This is important because at lower pH levels, the Hb is in its T state; therefore, causing Hb to not bind with oxygen

Know the Koshland (SN2) mechanism for HEWL. What makes the alkoxide a good leaving group in HEWL?

The poor alkoxide leaving group is protonated by E35 to form an alcohol.

What is limits the rate of an enzyme-catalyzed reaction? Be able to identify ES and the transition state on a free energy plot.

The rate limiting step in enzyme catalyzed reactions is the reaction of the ES unit to the products. The reactant reaction with the enzyme to form the ES unit happens very quickly due to a lower activation energy that helps enzymes act as a fish ladder. However, once the ES complex is formed, the activation energy to form the products is greater making the reaction take more time.

What is meant by a thermodynamic pit? Do enzymes bind substrate 'tightly'?

Tight binding of a substrate prevents S from reaching the transition state. If ES binds the substrate tightly, it stabilizes the ground state or strained conformation of substrate causing the reaction coordinate to move further from the transition state.

Know how the Hb and the carbonate buffer system work together in the body and the lungs (relate to Le Chatelier's Principle) to facilitate gas exchange. Name two ways that Hb helps get rid of CO2 in the body.

When Hb changes to the R-state, it is deprotonated and shifts the reaction to produce carbonic acid. Carbonic acid then decomposes to H2O and CO2. Some of the CO2 will be released through diffusion and others will react with Hb to produce carbamates. This formation can be reversed in the lungs with the release of 2,3-BPG and CO2 when you exhale. To get rid of CO2 in the body, it generally shifts H2O + CO2 H2CO3. This shifts the equilibrium and produces bicarbonate. The deprotonation will be picked up by Hb and will convert it to the protonated Hb state (T-state). When protonated, the Hb will bind to oxygen, releasing the proton and shifting the equilibrium to produce CO2, which is exhaled out.