Hb and Mb
What is the quaternary structure of
Myoglobin?
Hemoglobin?
None or monomer
Tetramer (2 alpha and 2 beta)
Enzymes will affect this (1) to speed up the rate of the reaction but it will NOT affect this! (2)
(1) Activation Energy
(2) Delta G of the reaction
If the Km increase how does that affect affinity?
Given the following values what is the Km and Vmax of this enzyme?
Y-int = 4
X-int = - 0.06
Vmax = 1/Y-int = 1/4 = 0.25
Km = -1/X-int = 16.667
Please be able to get the X and Y intercept from an LB plot!
What is the difference between homotropic and heterotopic effectors, and what is an example of each?
Homotropic effectors are present either a reactant or product in the reaction such as Oxygen.
Heterotopic effectors are not present as reactants or products such as Cl or 2-3BPG etc.
His E7
What does His F8, Val E11 and Phe CD1 do?
This is the apoenzyme plus it's cofactor
The Holoenzyme (Think "W"hole enzyme)
Describe the X-intercept and Y-intercept on an LB plot
X-int: -1/Km
Y-int: 1/Vmax
Calculate the initial velocity of an enzyme given the following values:
Vmax = 12 M/s
Km = 4.5 M
[S] = 8 M
(Please be able to get Km and Vmax values from an LB plot!)
Vo = Vmax * [S] / Km + [S] = 7.68 M/s
What substitution takes place and where?
What state does it polymerize in?
Sickled RBCs will polymerize in the T state!
Hydrogen and bicarb will bind to these things that make the T stat "tense"
H-Bonds and Ion pairs only present in the T State
What type of catalysis is involved in the RNAs A mechanism?
GABC
His 119: GB then GA
His 12: GA then GB
If the Vmax does not change when an inhibitor is added what type of inhibitor is it?
Competitive Inhibitor
Given the pO2 of myoglobin is 46, what is the fractional saturation (YO2)?
YO2 = pO2/ (P50 + pO2)
YO2 = 46/ (2.8 + 46)
YO2 = 46/(48.8)
YO2 = 0.943
What is the shape/characteristic of the binding pocket for
Trypsin
Chymotripsin
And Elastase
Trypsin: Deep and negatively charged Asp189
Chymotripsin: Deep and hydrophobic
Elastase: Shallow and Uncharged
At a high pH the hemoglobin in your body will favor this state.
R State (high pH = low H+ concentration less of an inhibitor means the protein is active)
Bonus questions where does the H bind?
Describe the Serine Protease Mechanism
1) GABC via His 57
2) Covalent Ca. (Ser 195)
3) Electrostatic stabilization (Asp 102 and the 2 backbone amid NH's)
Be very familiar with this and prepared to answer several MCQ's on the exam about this very important mechanism.
What does DIPF inhibit? And what type of inhibitor is it?
It will inhibit all of the enzymes with a serine proteas in the active site. It is a group-specific reagent. For example it will inhibit the following: Serine proteases such as trypsin, chymotrypsin, elastase, thrombin, AChE, and so on.
What is the turnover number for a particular enzyme given the following information:
Km = 4.5
Vmax = 8.3
[ET] = 200
Turnover Number = Kcat = Vmax / ET
So: Kcat = 8.3/200 = 0.0415
CO2 + Pyruvate + ATP -----> Puryvate-CO2 + ADP + Pi + H+
What is the EC Classification for this reaction?
EC 6 Ligase
The hill coefficients below mean this:
3?
.4?
0?
Positive cooperative (ex. Hemoglobin)
Negative cooperative
Non-cooperative (Ex. Myoglobin)
What are the EC classification numbers and what do they each mean?
EC Numbers:
1 - Oxidoreductase
2 - Transferase
3 - Hydrolase
4- Lyase
5 - Isomerase
6 - Ligase
What does Saquinavir inhibit?
HIV Protease Mechanism
X -int: -3.5
Y-int: 6.3
X-int' : -3.5
Y-int' : 8.4
[I] = 100 mM
(Please be able to determine these values from an LB plot)
alpha = Vmax/Vmax'
aplpha = 1 + [I]/Ki
so: Vmax/Vmax' = 1+ [I]/Ki
Solve [I]/Ki = Vmax/Vmax' - 1
And Ki = [I] / (Vmax/Vmax' - 1)
The Perutz mechanism is important for the exam. What are the nine steps that you need to know?
1) T-state is locked in this “tense” conformation by H- bonds & ion pairs that are not present in the R-state!
2) It is difficult for the T-state to bind the 1st O2: In T-State, His E7 & Val E11 block the 1st O2’s access to heme:
3)In T-state (deoxy state) the Fe2+ iron is ~0.6 Å out of the heme plane
4)Eventually, when 1st O2 binds it pulls Fe2+ back into the heme plane.
5)Since the His F8 is attached to the Fe2+ it is pulled towards the heme plane
6)Because the His F8 is part of Hb’s F helix, it pulls the F helix like a lever on a fulcrum- F helix translates (moves) 1Å
7)When the F helix moves, to accommodate the spatial rearrangement caused by its movement, ab pairs rotate 15o with respect to each other (= change in 40 structure)
8)In R-State, the Tà R shift in 4o strO2 has clear access to the heme structure causes His E7 & Val E11 to move out of way = on the other 3 subunits that have not yet bound O2!
9)The energy in the formation of the Fe-O2 bond formation drives the Tà R transition!