Biochem chapter 5
What is covalent catalysis?
-Occurs when part or all of a substrate molecule forms a covalent bond bond with a component of the enzyme's active site before transfer to a second substrate; will stabilize an intermediate in a chemical reaction by covalently attaching to one part of the substrate -when a nucleophilic in the active site forms a transient covalent ES complex -In the presence of a covalent catalyst (an enzyme with a nucleophilic group X:) the reaction becomes(it is two steps btw, unlike other catalytic reactions): A-B + X: --> A-X + B --> A + X: + B Note: the one in the middle is the covalent intermediate -covalent catalysis uses covalent bonds two connect the substrate to the active site on the enzyme, when other catalytic methods just use weak intermolecular forces -REMEMBER! For all enzymes, the free enzyme must be regenerated in order for it to be a true catalyst! -Polar amino acids like Ser are good nucleophiles because they have free electron pairs, but Ser becomes a better nucleophile when its H is removed. -All polar amino acids with lone e:- are good nucleophiles, like Cys, Ser, he mentioned His too...(maybe when it is deprotonated?), and etc.)
How does metal ion catalysis work?
-metal ion is involved in the reaction -protein binding to metal, for example zinc, zinc can then be involved in reaction stuff. -ionic interactions between an enzyme bound metal and a substrate can help orient the substrate for reaction or stabilize charged reaction transition states -metals can also mediate oxidation-reduction reactions by reversible changes in the metal ion's oxidation state
What are the 6 ways enzymes decrease activation energy?
-transition state stabilized(from substrate binding) -entropy reduction(from substrate binding) -desolvation(from substrate binding) -general acid-base catalysis(but there is also specific acid-base catalysis, btw) -covalent catalysis -metal-ion catalysis
What is a spontaneous reaction?
-ΔG (from ΔG=ΔH-ΔTS, or in a reaction coordinate diagram, when Gp-Gr=-ΔG) The pic to the right is a very helpful picture showing a spontaneous reaction, although it says exothermic instead of exergonic. He wants it to be specifically exergonic, not exothermic, because exothermic means heat is released but exergonic means release of energy. He wants us to remember EXERGONIC reactions are spontaneous, because exergonic refers to ΔG but exothermic refers to ΔH and we are talking about ΔG. (Also, replace the y coordinate with G, so kJ/mol and that is what we are looking for, and the ΔH would be ΔG)(also where it says activated complex is where the transition state would be). Spontaneous reactions don't have to have energy put into them in order to occur, because they are favorable and actually they release energy.
What are the units for partial pressure of O2 (pO2)?
101KPa=1ATM=760Torr=760mmHg
What order of reaction is S <=> P ?
1st order
What is induced fit?
A change in shape of a protein (includes enzymes and substrates, too, btw) that occurs when the ligand binds to the protein. This conformational change of the protein causes the binding site to be more complementary to the ligand so that the ligand fits better and is held more tightly.
What is a coordinate bond?
A covalent bond in which both electrons in the shared pair come from the same atom. In the coordinate bonds between Fe2+ and nitrogen, nitrogen is the one to donate both of its electrons.
What does a protein bind to and where?
A ligand binds to a protein at the protein's active site
Where does substrate bind on an enzyme?
A substrate binds on the enzyme at the enzyme's active site.
An enzyme is a specific kind of protein. What does an enzyme bind to and where?
A substrate binds to an enzyme at the enzyme's active site.
Are oxygen and H+ bound on the same spot of hemoglobin?
As we saw, CO2 binds at the amino terminal end of each subunit. Like CO2, H+ does not bind in the same place as O2 and CO2, but it also does not bind in the same place as CO2 either. Where it binds does help stabilize the T state like CO2 does when it carbamylates, though, too. (Note: I am not saying here that H+ carbamylates). H+ binds to several amino acids. A big contributor to the Bohr effect is a histidine(His^146, or called His HC3) of the β subunits. When it is protonated, this His forms and ion pair with an Asp(Asp^94, or Asp FG1) which stabilizes the T state. So more H+ = lower pH = more protonated His = T state stabilized = more Hb in the T state so that where there is a lot of H+, more O2 can be delivered. Again, this is the bohr effect. Also, CO2 does carbamylation which stabilizes the T state, so also, more CO2 means more Hb in the T state because it carbamylates as CO2 and because it can also undergo a reaction that creates more H+(and bicarbonate, but the CO2 is gone/gotten rid of/hydrated.
When does the body make more BPG?
At high altitudes, oxygen concentration decreases, so more BPG is found in the blood at high altitudes so the body has the partial pressure of oxygen in its tissues that it needs/so body is delivered enough O2. So higher elevation = partial pressure of oxygen lower than usual so T state needs to be stabilized more in order to release O2.
In the transition state, what is the likelihood of the forward reaction vs the reverse reaction?
At the transition state, there is a 50/50 chance of the substrate doing the forward or the reverse reaction
Why does Mb have a lower Kd(P50) than Hb?
Because Mb is for oxygen storing, so you want it to have a lower Kd because you want there to be less [P] and [L]. Mb is for storing, so want the protein to be in high-affinity state all of the time. Want mainly only [PL]. At θ=0.5, want most of the ligand bound. Mb most of the time in R-state, high affinity. Hb fluctuates between high affinity state R and low affinity state T. So Mb is better/more efficient at O2 binding compared to Hb which is good because Mb is for storage so it wants to be in high affinity state all the time and hold onto the O2. Hb is for O2 transport, so it wants to let go of O2 sometimes so it sometimes want to hold onto O2, sometimes wants to let go of it.
What is cooperative binding and allosteric binding, both terms referring to oxygen binding in hemoglobin?
Binding can be considered "cooperative" if the binding of the first molecule of B to A changes the binding affinity of the second B molecule, making it more or less likely to bind. In other words, the binding of B molecules to the different sites on A do not constitute mutually independent events. In cooperative binding, when the second ligand molecule binds, it does so at a higher affinity than the first ligand binding to the protein which leads to positive cooperativity. Allosteric enzymes are enzymes that change their conformational ensemble upon binding of an effector, which results in an apparent change in binding affinity at a different ligand binding site. (Note: myoglobin and hemoglobin are not enzymes, they are proteins that bind to ligands. Enzymes bind to substrates, btw). In book, it mentions that an allosteric protein is one in which the binding of a ligand to one site on a protein affects the binding properties of another site on the same protein.
How does catalysis by entropy (disorder) reduction work?
Catalysis in itself causes entropy reduction because substrate and enzyme have to be by each other and have to line up correctly for weak intermolecular forces to occur and for each enzyme to do its thing with the substrate to bind(so less entropy). Also, look at picture to the right. The first reaction shows two molecules becoming one. There is not rate enhancement yet because there has not been any decrease in entropy yet, so rate enhancement = 1. The second reaction has the products of the first reaction as its reactant. Through the first reaction, the two molecules have become one. This causes a reduction in entropy so the rate enhancement is 10^5. In this reaction, a ring is formed and this also reduces entropy. This ring is used as the reactant in the third reaction. Because it is a ring, entropy has been reduced so the reaction is 10^8 times faster. Enzymes also reduce entropy and so activation energy these ways, by bringing molecules together and creating rings so that reactions can occur faster. Entropy is also reduced by lining molecules up so that more productive reactions occur. So, enzymes line up stuff. In the third reaction, btw, entropy is also reduced because since some bonds are made and another ring is made, free rotation/bond rotation is not allowed to occur anymore which also reduces entropy/disorder and so decreases activation energy. (picture and explanations also on p.196 in Figure 6-7).
What do catalysts do?
Catalysts lower the activation energy of a chemical reaction, so the transition state is made lower. Catalysts do not effect the equilibrium of a reaction, so they do not effect if a reaction is spontaneous and they do not effect ΔG.
What types of reactions are catalyzed by hydrolases?
Catalyze hydrolysis reactions (transfer of functional groups to water); catalyze reactions involving water (often reactions that split bonds - I believe this is called hydrolysis)
What do enzymes do?
Catalyze reactions, lower activation energy, speed up reactions
What types of reactions are catalyzed by lyases?
Catalyze reactions, often elimination reactions. Cleavage of C-C, C-O, C-N, or other bonds by elimination, leaving double bonds or rings, or addition of groups to double bonds(so I think the reverse reaction(reverse of elimination) can also occur) -In the reactions catalyzed by lyases, remember that lyases don't use hydrolysis or oxidation. That is left for other enzymes, hydrolases and oxidoreductases.
What types of reactions are catalyzed by oxidoreductases?
Catalyze transfer of electrons (hydride ions or H atoms)
What types of reactions are catalyzed by transferases?
Catalyze transfer of various functional groups (Example: kinases)
What types of reactions are catalyzed by ligases?
Creation of bonds(example:DNA ligases). Formation of C-C, C-S, C-O, and C-N bonds by condensation reactions(H2O=by-product of condensation reactions) coupled to cleavage of ATP or similar cofactor
What is the general reaction for an enzyme?
E + S <=> ES <=> EP <=> E + P
What is the general reaction for enzyme and a substrate?
E+S <=> ES <=> EP <=> E+P E=enzyme S=substrate Graph to the right is very helpful. Look at it!
Are enzymes complementary to the ES complex or the the transition state?
Enzymes are complementary to the (highest) transition state. Interactions between enzyme and substrate, which are mainly weak intermolecular interactions, are maximized at the transition state. This is how enzymes get substrate to reach their transition and so how they lower the activation energy. Look at very helpful stickase picture to the right. It also shows why enzymes do not maximize interactions, mainly weak intermolecular interactions, for the ES complex. If it did this, the substrate would be held in the active site of the enzyme so tightly that it would make it even harder than without any enzyme to reach the transition state. The ES intermediate would be stabilized, which is kind of almost the opposite of what we want to happen...we want it to reach an unstable transition state, where both the forward and reverse reaction are likely to happen(transition state = very unstable state).
What are enzymes?
Enzymes are normally proteins that catalyze reactions (some are not proteins, like RNA). They are very specific on the reactions they catalyze, and they catalyze reactions by speeding them up. They speed up reactions by lowering the activation energy.
What does the reaction coordinate diagram on the right show?
Enzymes stabilize stuff by lowering the activation energy. They do this by forming mainly weak intermolecular interactions between the enzyme and the substrate and stabilize the transition state and so lower the activation energy.
CO2 + H2O <=> H2CO3 <=> H+ + HCO3- What is happening here/what is the story of this?
Example: Workout, muscles use oxygen, oxygen decreasing in tissues, CO2 concentration increases, then: CO2 + H2O <=> H2CO3 <=> H+ + HCO3- So, H+ is being produced, higher proton concentration = lower pH = T state stabilized = oxygen being delivered better to muscles
Is CO or O2 better at binding to Fe2+ in heme?
Fe2+ in heme binds better to CO because the bond is straighter/more favorable.
Can use myoglobin equations for hemoglobin?
Figure out.
For myoglobin, y=θ=?
For myoglobin, Y=θ=binding sites occupied/total binding sites=[PL]/[PL]+[P]=[unbound ligand]/[unbound ligand]+dissociation constant=[L]/[L]+Kd. Also, θ=Y=fractional binding
How many coordinate bonds can the Fe2+ make and to what are they made?
Four of the coordinate bonds are between the Fe2+ and the four nitrogens of the prosthetic group, 1 is between the proximal His F8(the 8th residue/amino acid of the alpha helix F) and the Fe2+, and one is between the Fe2+ and the O2(diatomic oxygen) which binds to hemoglobin and myoglobin
How is is CO2 in the body related to H+?
H+ and CO2 are both by-products of cellular respiration that the body needs to be able to get rid of. They are taken from the tissues and excreted through the lungs and kidneys.
How is the binding of H+ and CO2 related to the binding of O2 in hemoglobin?
H+ and CO2 bind to hemoglobin is inversely related to O2 binding in hemoglobin. So where a lot of H+ and CO2 is located and so bound to the hemoglobin, not a lot of O2 will bind and so hemoglobin will be in the T state. This means, as pH decreases, the amount of hemoglobin in the T state will increase. This means in a graph of θ vs pO2, the lower the pH, the more shifted to the right the graph will be because as pH decreases, more H+ will cause less fractional binding of O2 as amount of T state hemoglobin increases. So: HHb+ + O2 <=> HbO2 + H+ So, H+ and CO2 bind to hemoglobin inversely related to O2 binding in hemoglobin. So where a lot of H+ and CO2 is located and so bound to the hemoglobin, not a lot of O2 will bind and the hemoglobin will be in the T state. This would be in places like the tissues where the pO2(concentration of O2) is low but the pCO2 and the concentration of H+ is high. Inversely, in places where the oxygen concentration/pO2 is high like in the lungs, hemoglobin binds O2 and releases H+. So in places of high pH, meaning low concentration of H+, hemoglobin is mainly in the R state(high affinity state, so it will pick up O2). This is called the bohr effect. Look on p.170 at figure 5-16 for a good graph probs will be on the test.
What does a heme group consist of?
Heme consists of a porphyrin ring system, and either an Fe2+ or an Fe3+ in the middle of that porphyrin ring system
Does myoglobin or hemoglobin bind to oxygen and act as a delivering protein?
Hemoglobin binds to oxygen and delivers it whereas myoglobin binds to oxygen and stores it
What must hemoglobin be able to do in order to do its job and deliver oxygen effectively?
Hemoglobin must be able to bind oxygen effectively in the lungs, where the pO2 is about 13.3 kPa, and release oxygen in the tissues, where the pO2 is about 4kPa.
What does the disease sickle cell anemia show?
How important amino acid sequence is for determining the secondary, tertiary, and quaternary structures of globular proteins, and so their biological functions. HbS=hemoglobin from sickle cells When hemoglobin from sickle cells is deoxygenated, it becomes insoluble and forms polymers that aggregate into tubular fibers. Normal Hb stays soluble. This occurs because in both beta chains, their is an amino acid substitution of Val instead of Glu (so goes from being charged to not charged - not charged = hydrophobic = aggregates because now on the Hb there is a sticky hydrophobic spot that will interact with other Hb that have these sticky, hydrophobic spots). These long aggregated rods cause the sickle cell shape. This causes serious medical conditions, and I am guessing its interactions with O2 are messed up. For more info. on sickle cell, can look on sapling chapter 5 homework, #11. (If have time, add more from that onto here but fyi, did not talk about in class and is kind of complicated so might be learning a complicated thing for no reason --> so just read through it or write in here about it if really have free time to write about it which is doubtful...)
Why must the heme be bound to protein?
If heme is a free molecule without protein, one O2 molecule can bind to two free heme molecules(or two Fe2+). This can cause Fe2+ to be permanently oxidized into Fe3+. Fe3+ is unable to bind to O2 so it would be a useless heme and it would be dangerous if all heme were without protein, then.
What if the body produces no BPG?
If the body produces no BPG, then the line will be a hyperbola on a graph of fractional binding vs pO2 because the T state will not really occur. So, really we need BPG to stay in the T state.
What is the shape of a myoglobin curve of θ vs pO2 in comparison to a hemoglobin curve of θ vs pO2?
In a graph of θ vs pO2, a myoglobin curve is hyperbola shaped vs a hemoglobin curve which would be in an S shaped, sigmoidal curve due to the cooperative binding and one subunit affecting another subunit. Example: hemoglobin is composed of four subunits that are each individually very similar to myoglobin. When one of the subunits binds to an O2, that bound subunit affects another subunit by making that other subunit likely more to bind. This is cooperative binding. It leads to a sigmoidal shaped curve as the molecule slowly goes from a low affinity state to a higher affinity state with more ligand bound, so a higher value for θ.
In fetal hemoglobin, what type of subunits are found? What does this cause?
In fetus, beta(β) subunits are not found. Alpha(α) and gamma(γ) subunits are found in fetal hemoglobin. This is because the fetus gets all of its oxygen from its mothers blood, so it must be able to hold onto oxygen better than mom. In other words, the R state must be more favored than the T state in a fetus. It does this through the gamma subunits, which with the alpha subunits creates a tetramer that has a much lower affinity for BPG than a tetramer made up of beta and alpha subunits. Since the BPG stabilizes the T state, a loss of affinity for BPG by hemoglobin will cause the R state to be more stabilized and so the fetus will hold onto the O2 better than mom.
For 1st order reactions, what does the rate of the reaction depend on?
In first order reactions, the rate of the reaction is dependent only on the one reactant(the substrate).
What is general acid-base catalysis and what is specific acid-base catalysis?
In specific acid-base catalysis, H+ is transferred and only the H+(or H3O+) or OH- ions present in water are used (so it involves H2O). General acid-base catalysis refers to proton transfer mediated by weak acids and bases other than water, so HA or B-. General acid base catalysis occurs with His a lot because it can protonated or not protonated pretty near to the physiological pH.
Why is CO dangerous?
It binds to Fe2+ in heme way better than O2. CO has a 20,000-fold stronger binding affinity in myoglobin compared to O2.
How does ΔG affect the rate of a reaction?
It does not. The change in free energy only tells if the reaction is favorable or not, and so if it will happen. For example, -680kJ/mol is the ΔG of paper turning into CO2 and H2O(the oxidation of paper), so it is super favorable/spontaneous/exergonic. The only problem is that this reaction has a very large activation energy, so it will take a really long time for this to happen because it will take a long time for paper to get all of the activation energy it needs to reach transition state. Also, it has a high transition state because it has a high activation energy(goes from the substrate energy to the transition state). So, for paper, something like heat or some other kind of added energy will make the reaction happen faster but since it is exergonic, it is not needed for the reaction to occur.
What are the characteristics of a porphyrin ring system?
It is a conjugated system, planar, has four nitrogens in the middle of it, in the center of the four nitrogens is an Fe which can either be an Fe2+ or an Fe3+
What is Kd for myoglobin?
Kd=big Kd=dissociation constant=[P][L]/[PL]=[products]/[reactants]=dissociation rate constant/association rate constant=rate of reverse reaction/rate of forward reaction=kd/ka=little kd/little ka, from PL <=> P+L. Also, Kd=big Kd=1/Ka=1/big Ka=1/association constant
What is oxidation and reduction?
LEO the lion says GER (Lose Electrons:Oxidation. Gain Electrons:Reduction) The reducing agent loses electrons and thus is oxidized in reaction. The oxidizing agent gains electrons and thus is reduced in the action. Reduction can also refer to losing oxygen and oxidation then would be gaining oxygen.
What is the greek alphabet?
Look at pic to the right(click).
How many helices does myoglobin have?
Myoglobin has 8 alpha helice segments, A-H
What are some of the main similarities differences between Mb and Hb?
Myoglobin(Mb) is for oxygen storage whereas hemoglobin(Hb) is for oxygen transport. Mb is a monomer made of 8 alpha helices. It has tertiary structure as a globular protein. It contains a heme prosthetic group that has four nitrogens, a conjugated porphyrin ring system, and an Fe2+ iron right in the center of the planar/flat/plane-like ring system. Distal His(His^64, or His E7 in myoglobin) helps to hold the O2 in place and make its binding more favorable. Proximal His is His^93 in myoglobin, or also called His F8, and it is connected to the Fe2+. Hb is a tetramer composed of two alpha subunits and two beta subunits. All four subunits are each composed of eight alpha helices, like in myoglobin. In fact, each singular subunit is like a myoglobin. Like myoglobin, each subunit has a heme prosthetic group. The heme prosthetic group also interacts with a histidine. Hb also has the E and F helices with the His on them, one distal and one proximal, and they both function the same as the ones Mb, but they have different numbers. For instance, for Mb, Hbα, and Hbβ, the distal His residue is His E7 for all structures, but corresponds to His^64, His^58, and His^63 in the linear sequence of Mb, Hbα, and Hbβ, respectively. For all three, the proximal His residue is F8. Also, Hb makes it all the way up to quaternary structure. One thing that is different between heme from Mb and heme from Hb is that in Mb, the Fe2+ is right in the middle of the plane, but in the Hb, the Fe2+ is not in the middle of the plane. The iron is actually puckered. To be more clear, hemoglobin switches between two stable conformations because hemoglobin has protein to protein interactions due to being in quaternary structure. The two stable conformations are T state/taught state/puckered and R state/relaxed/not puckered.
Is the R state likely to release oxygen?
No, in fact, it is more likely to actually TAKE O2 out of cells
Is a reaction intermediate the same as a transition state?
No. A reaction intermediate actually has a finite lifetime, which is anything longer than 10^-13, a bond vibration.
What is a nucleophile?
Nucleophile is a chemical species that donates an electron pair to an electrophile to form a chemical bond in relation to a reaction. All molecules or ions with a free pair of electrons or at least one pi bond can act as nucleophiles. Because nucleophiles donate electrons, they are by definition Lewis bases.
Draw a porphyrin ring with Fe2+
OK
Draw out amino acids from memory and draw out a peptide to make sure still know how to do peptide bonds and stuff.
OK
For the rest of this, review notes and book because it is a bunch of equations and graphs and reactions on enzymes and then on inhibitors. So review notes and after test put notes in order so can study them and this and powerpoints and outlines for final. In book, enzyme kinetics from p. 199-203. Important inhibition stuff from p.207-210. Good summary on all of this from 212-213. Need to make sure have important stuff like ions pairs that stabilize the T state, what the Kd is and how to find it, all the amino acids, how enzymes catalyze stuff, the types of enzymes there are, all the equations and reactions on enzymes including inhibitor stuff, chemotrysin stuff, enzyme regulation stuff, and etc memorized before the test. Also look over sapling, and also look over both quizzes if both are available(you have one!). Also look over these quizlet cards. Also look over notes in book for chapter 6 enzyme kinetics and inhibitors. Figure out chemotrypsin and enzyme regulation stuff, too, by looking at notes and book and maybe some videos on youtube or kahn academy because don't get. If have time, do some sapling problems. Also review how to draw out a peptide and do peptide bonds between amino acids. Also take a peak over powerpoint slides and if have time, class outlines.
OK
Get graphing calculator for test.
OK
What is BPG and what does it do?
Ok, so as we talked about before, the T state is stabilized by specific ions pairs, and in the T state, there is a pocket between the β subunits that is created by these interactions between these ions. As the T state transitions to R state, the space between the β subunits narrows and the ion pairs that stabilize the T state are broken. BPG is a molecule that stabilizes the T state. BPG has a lot of negative charges(it is similar to diglyceraldehyde with a lot of neg. charged phosphate groups), so the binding site of BPG on hemoglobin is this cavity/pocket between the β subunits in the T state because this pocket is lined with a lot of positive charges. Because the BPG is in this pocket, the pocket can not narrow and so the hemoglobin cannot transition from T state to R state. So in the end, BPG lower's hemoglobins affinity for oxygen by stabilizing the T state. Because of this, in a fractional binding vs. pO2 graph, the sigmoidal line will be shifted more to the right. Also, BPG is a heterotropic effector, meaning it is a molecule that effects the affinity of the protein, but the molecule is not the ligand. Homotropic effectors effect the affinity of the protein, but they are the ligand(in this case, O2).
On a graph of θ vs pO2 (or [L]), where is there an asymptote?
On the y-axis there is an asymptote at 1. θ=1 at an infinite concentration of ligand, so can't really have full binding.
What are the 6 types of enzymes?
Oxidoreductases, transferases, hydrolases, lyases, isomerases, and ligases.
Hemoglobin is cooperative, so if an O2 binds, it will go from T to R state. (So, what really controls what state/conformation a hemoglobin is in?)
Partial pressures control what conformation the hemoglobin is in. In the lungs, where the partial pressure of O2 is high, hemoglobin will be in the R state so that it can pick up the O2. In the tissues, where the partial pressure of O2 is low, the hemoglobin will be in the T state so that it will deliver that O2 to the tissues/let that O2 go to the tissues instead of bind to it(T state = low affinity). So really, fractional binding of hemoglobin is really dependent on the partial pressure of O2 in the body. High partial pressure = R state, low partial pressure = T state. Look at figure 5-12 on p. 166 for a really good graph of how this works and the curve for hemoglobin in high affinity state, a curve for low, and then the true curve for hemoglobin which is inbetween them because it is a sigmoid curve that transitions first from low affinity state in the tissues where the partial pressure is low, to high affinity in the lungs where partial pressure is high). NOTE: Partial pressures work with the cooperative binding of hemoglobin! This is because, at higher pressures, logically, O2 is more likely to bind to the hemoglobin. So, if the pressure is higher, O2 will bind to the hemoglobin. If the hemoglobin was in T state, it will then slowing go from T state to R state and so the hemoglobin will be at high affinity R state where the pressure is high. Basically, the high pressure, through making the O2 bind to the hemoglobin, signals to the molecule that it needs to be in R state. SO, the binding of an O2 causes this conformation change to occur and the high pressure is what drives the O2 to bind. At low partial pressures, way less O2 will bind. SO, low partial pressures signal to the molecules that there is less O2 around and it needs to be in low affinity state.
R=?
R=gas constant=8.314Joules/mol*K (K=temp. in kelvins here, not Kd or Keq or little k, which is the rate constant)
When is there no rate enhancement due to entropy?
Rate enhancement = 1 = no rate enhancement
k1 For S <=> P, rate=? k-1 (k subscript 1, not negative 1)
Rate only depends on the reactants, so rate=k1[S]
If a reaction depends on two different molecular compounds(we did not really learn about this, btw), what is the rate law?
Rate=V=k[S1][S2]=M^-1/S^-1
Which animals have a lot of myoglobin?
Sea diving animals such as whales have a lot of myoglobin for oxygen storage
Why does the iron sometimes pucker in hemoglobin?
So, hemoglobin has two stable conformations. They are the R state and the T state. Oxygen can bind to either state, but it has a significantly higher affinity for the R state. So the T state is the predominant conformation of deoxyhemoglobin. The T state is tense and the R state is relaxed because the T state is stabilized by a greater number of ion pairs which are at the α1β2 and α2β1 interfaces. When O2 binds to hemoglobin in the T state, the hemoglobin undergoes a change in conformation into the R state because the R state has a way higher affinity for O2. When the whole hemoglobin protein undergoes this transition, the structures of the individual subunits change very little. The key is though, the αβ subunit pairs slide past each other and rotate, which causes the pocket between the β subunits to narrow. Because of this, in this transition as T changes to R, the pocket narrowing causes some of the ion pairs that stabilize the T state to break and new ones that stabilize the R state are formed(this, all in response to O2 binding). Also, as the T transitions to R, there is a change in the positions of key amino acids around the heme. So, in the T state, the porphyrin is slightly puckered, causing the heme iron to protrude slightly on the proximal His(His F8). The binding of O2 causes the heme to assume a more planar conformation, shifting the proximal His and the attached F helix. Note: these changes lead to adjustments in the ion pairs at the α1β2 interface, so it connects back to key ion pairs stabilizing the T state being broken and adjusted to R state ones. So, it is kind of like the T state has a bulging heme because the heme is like the rope in the middle of a tug of war. Without the O2, there is only one side of the rope being pulled, and it is being pulled by the proximal His F8. Because of this, the heme porphyrin ring is puckered on the His F8 side and the iron bulges out a bit on that side. In the R state, the heme porphyrin ring is not puckered and the iron does not bulge because both the O2 and the proximal His F8 both pull on the iron. So, in the R state, the heme is flat and the iron does not bulge.
-ΔG shows?
Spontaneous, favorable reaction
Substrate binding is the first thing that happens in enzyme catalysis, and it lowers activation in three ways. What are these three ways?
Substrate binding helps to lower activation energy by: -Transition state stabilization(not ES complex stabilization!) -Entropy reduction(lining up molecules perfectly so that productive collisions occur is one way this occurs) -Desolvation = removal of solvent, usually water - when weak interactions occur, water is displaced - removal of water molecules makes the substrate molecule more reactive with the active site of the enzyme - need water to be removed because they get in the way and form H bonds between the substrate and the water - need desolvation so that water is gone and so that weak interactions between the solvent and the enzyme are maximized because this is when the transition state of the substrate is reached --> so the weak intermolecular forces between the substrate and the enzymes active site stabilize the transition state/help the substrate to reach its transition state
How can a hyperbola graph be turned into a linear, straight line?
Take the reciprocal. For ligand and protein(from chapter 5, if the graph of θ vs [L] is turned to 1/θ vs 1/[L], then the slope would be Kd. θ=[L]/(Kd + [L]), and this creates the hyperbola (the equation for the hyperbola). If you take its reciprocal, it can be seen that 1/θ=1/([L]/(Kd + [L])) eventually comes out to be 1/θ=Kd(1/[L])+1, which is essentially y=mx+b. (this is actually a chapter 5 thing).
In hemoglobin, why is the T state more tense and the R state more relaxed?
The T state is more tense because, even though the R state has some specific ones for it too, the T state has a lot of ion pairs that stabilize it (Figure 5-9, p.164).
What are the main ion pairs that stabilize the T state of deoxyhemoglobin?
The T state is tense and the R state is relaxed because the T state is stabilized by a greater number of ion pairs which are at the α1β2 and α2β1 interfaces. In deoxyhemoglobin in the T state, there are interactions between ion pairs which keep it in a tenser state than R. Here are some of the main ion pair interactions which keep T stabilized: On the β subunits, Asp- and His+ (Asp FG1 and His HC3) interact. Lys+ of the α(there are two α subunits so this happens twice, btw) interacts with the α-carboxyl group(basically, the carboxyl terminal) of the His+ on the β subunit(so Lys C5 of α1β2 subunit interacts with His C3's carboxyl terminal end on the β subunit. Also, Arg HC3(Arg+) on one α subunit interacts with Asp H9(Asp-) on another α subunit. Again, this happens twice. Also, on the α subunit, the amino terminal end of Lys C5(Lys is positively charged, and its amino terminal end is too, but its R group is interacting with His+/His HC3(on a β subunit) at its negatively charged carboxyl terminal so only the amino terminal end is left to interact) interacts with the carboxyl terminal end(negatively charged) of Arg HC3 on another α subunit. Again, this happens twice. Also, Arg HC3 has a positively charged R group that again is interacting with Asp H9(Asp-, negative R group). They are on different α subunits and this again happens twice. Look on p. 165 at Figure 5-9 for more information and a really good visual to these words.
How can you find Kd(P50 when using partial pressures) for myoglobin on a graph of θ vs [L] (θ vs pO2 (kPa) for O2 as a ligand because O2 is a gas so need to use partial pressures for concentration of ligand with the units in kPa)?
The [L] or pO2 at which half of the available ligand binding sites are occupied corresponds to Kd or 1/Ka since Kd=1/Ka. So at θ=0.5, look at the pO2 or the [L] and that is the Kd(Kd in units of M). Look at pic because it is really helpful(to the right).
What triggers a hemoglobin in T state to turn to R state?
The binding of O2 to a hemoglobin subunit in the T state triggers a change in conformation to R state.
What does the body do since CO2 is not very soluble in aqueous solution? What does the body do with this excess CO2?
The body hydrates most of the CO2: CO2 + H2O <=> H2CO3 <=> H+ + HCO3- So, as we can see, this is another source of H+. Hemoglobin transports 40% of total H+ and around 15-20% of CO2 formed from the tissues to the lungs and kidneys. The remainder of the H+ is absorbed by the plasma's bicarbonate buffer that we talked about in chapter 2. The remainder of the CO2 is transported as dissolved HCO3- and CO2. One way that hemoglobin transports CO2 is through carbamylation of the amino terminal end of the hemoglobin(so the CO2 does not connect to the hemoglobin in the same place that O2 and CO do). So, in carbamylation, since there are four subunits in hemoglobin which are four globin chains, four CO2 can be transported to the lungs. This is carbamylation: O || CO2(aq) + +H3N-Hb <=> -O-C-N-Hb | H As we can see from this reaction, the positively charged amino terminal end loses two of its hydrogens in order for the O=C=O (CO2) to attach to the amino terminal end of one of the subunits of the Hb protein. So, more H+ is produced by this process as well in addition to the ones discussed above. In carbamylation, we can see that a positive charge from the amino terminal end is converted to a negative charge. This strengthens ionic bonds which further stabilizes the T state. This promotes the release of O2. In other words, in areas like the tissues where there is higher CO2 and lower O2, the hemoglobin is encouraged to release oxygen. So, in the T state, O2 is released and also CO2 stabilizes the T state and since it is attached to the hemoglobin, it can be transported where it will be excreted, for example in the lungs where CO2 partial pressure is low. CO2 partial pressure is high in the tissues, btw.
For the hemoglobin curve, when does it shift right?
The curve shifts right when there is less affinity for O2. When this occurs, O2 is released to the tissues at higher partial pressures than usual.
CO has a 20,000-fold stronger binding affinity in myoglobin compared to O2. What brings this number down to 40-fold for heme embedded in myoglobin?
The distal His(His^64, or His E7 in myoglobin(btw, proximal His is His^93 in myoglobin, or also called His F8)) holds the O2 in places. The Fe-O2 complex is much more polar than the Fe-CO complex. The Fe-O2 complex holds a partial negative charge and so is able to interact with the positive charge at the end of the distal His/His^64/His E7. So, the O2 is able to be held in place by the distal His/His^64/His E7 but the CO does not interact with the distal His/His^64/His E7 because it is not polar so it is not held in place any better. So the affinity of myoglobin for O2 is increased by about 500, but the affinity for CO is not changed therefore the 20,000-fold stronger binding affinity of CO for myoglobin compared to O2 is decreased to 40-fold.
What steps can be skipped in the general reaction for an enzyme and why?
The formation of EP can be taken out because we can assume this step is instantaneous. There is also the initial velocity assumption, which says that at the beginning there is no product present, so since [P]=0 at t=0, then k-2(the rate constant k with negative two subscript, not k minus 2) can be taken out. Also because of this, Vo=kcat[ES] (this is the equation for the rate law for formation of product), and the kcat in this is equal to k2, so kcat=k2.
What is a globin?
The globins are a widespread family of proteins, all having similar primary and tertiary structures. Myoglobin is a globin, it has 8 alpha helical segments from A-H. These segments are connected by bends such as AB, CD, and etc. Some bends have no amino acids, like BC.
What does a smaller Kd mean?
The ligand will bind tighter to the protein
What is k1 and k-1(k subscript negative 1, not k minus 1)?
The rate constants for the forward reaction and the reverse reaction of S <=> P -S=substrate -P=product -these are little k's, so they are rate constants, not equilibrium constants.
What if ΔG>0?
The reaction is not favorable, is not spontaneous, endergonic, products have a higher gibbs free energy than the substrate
What is a substrate?
The thing that binds to an enzyme (with just plain proteins, ligands bind) -the reactants
What does it mean if there is a high T to R ratio?
There is a lot more hemoglobin in the T state, which is the low affinity state, so this must be hemoglobin in a place where the partial pressure is low such as the tissues. The T state will be way less likely to bind to O2, btw.
Other than the protein portion of hemoglobin and myoglobin, what do both hemoglobin and myoglobin contain(hemoglobin with four and myoglobin with one)?
They contain a prosthetic group called heme
What is a transition state?
They do not actually exist because they are so short so their life is described as "infinite". Transition state is the moment when reaction is equally able to go either way, forwards or backwards reaction both equally likely to occur. It lasts around 10^-13 seconds which is basically just a bond vibration.
What types of reactions are catalyzed by isomerases?
Transfer of groups within molecules to yield isomeric forms(so just the rearrangement of molecules)
How can you tell when a reaction is at equilibrium?
Well, k1 and k2 are not equilibrium constants so you can't tell much about equilibrium from them, but when k1=k-1(k subscript negative 1, not minus 1), the reaction is at equilibrium
When concentration of H+ is high, how is the pH affected?
When H+ concentration is high, pH is low.
What are the units for a reaction coordinate diagram?
Y axis = free energy, G, in kJ/mol X axis = reaction coordinate which is just the stage in the reaction
Does it take energy for an enzyme to help a substrate reach its transition state (or continuing with the "stickase" example, does it take energy to get the stick to bend?)
Yes, it takes energy to get the stick to bend. Refer to pic on the right.
In a human, do you want a Fe2+ in your heme group or Fe3+?
You want Fe2+ because Fe3+ can't bind to oxygen
What is the sign for hemoglobin concentration?
[Hb]
What is the sign for myoglobin concentration?
[Mb]
What is a prosthetic group?
a non-protein compound permanently associated with a protein that contributes to the protein's function
What is a cofactor?
a substance (other than the substrate) whose presence is essential for the activity of an enzyme. Includes Mg2+, Fe2+, and etc. or a complex metalloorganic molecule called a coenzyme. Coenzymes act as transient carriers of specific functional groups. Usually we get them from our diet. A coenzyme or metal ion that is very tightly or even covalently bound to the enzyme is called a prosthetic group. Apoenzyme or apoprotein = protein part of enzyme. Complete, catalytically active enzyme = holoenzyme
What is catalase?
an enzyme that catalyzes the reduction of hydrogen peroxide The reduction of hydrogen peroxide reaction is: 2H2O2 <=> 2H2O + O2 H2O2=hydrogen peroxide This reaction does not show enzymatic activity, btw
What are the metric units
giga G 1000000000 10^9 mega M 1000000 10^6 kilo k 1000 10^3 hecto h 100 10^2 deca da 10 10^1 (none) (none) 1 10^0 deci d 0.1 10^−1 centi c 0.01 10^−2 milli m 0.001 10^−3 micro μ 0.000001 10^−6 nano n 0.000000001 10^−9 pico p 0.000000000001 10^−12 femto f 0.000 000 000 000 001 10^−15
Does myoglobin or hemoglobin bind to oxygen and act as a storage protein?
myoglobin
How can we tell if a reaction is spontaneous from Keq?
products are favored(the reaction is spontaneous) when Keq>1. Keq cannot be negative but it is opposite to ΔG in that higher numbers are spontaneous, not the lower ones.
How can you calculate rate enhancement due to an enzyme?
rate enhancement=kcat/kuncat=rate constant k of catalyzed reaction/rate constant k of uncatalyzed reaction
What are the units for k1?
s^-1, since rate is in M/s and [S] is the substrate concentration so it is in M
What does ΔG do vs activation energy?
ΔG is affected by the equilibrium constant, Keq (from S <=> P, where Keq=[P]/[S]). This can be seen in the equation ΔG=-RTlnKeq. In this equation ΔG is of course change in free energy, R is the gas constant R=8.314 Joules/mol*K, T is temp. in kelvins, and Keq as seen above is the equilibrium values of products over reactants. As can be seen through the equation, things like temp. can effect equilibrium. k affects the activation energy, so it affects where the transition state is and the rate of the reaction(from rate=k[S]). It does not affect equilibrium, Keq, ΔG, or anything telling about whether a reaction is spontaneous or not. Note: lowering the activation energy and the transition state will not make a reaction happen/will not make it spontaneous because the reaction is not favorable due to the products having a higher free energy than the substrate/reactants.
ΔG=?
ΔG=Gp-Gr=-RTlnKeq=kJ/mol or J/mol (J=joules)
In myoglobin, what value of fractional binding is almost reached since it is always in a high affinity state?
θ almost reaches 1, but not quite.
