Midterm 1

Big and hydrophobic amino acids are enriched in the protein core Van der waals - Transient & Weak - Fundamentally attractive - larger atoms = stronger interactions - strongly distance dependent
H+ Bond Donors & Acceptors (STNQ) Entropy measures the degrees of freedom:
Disulfide Bonds (C) in the Oxidized State - Stability depends upon the environment (Oxidizing vs. Reducing)
● lower entropy = more order - less H-bonding possibilities
H-Bonding (S, T, Y, N, Q, C, D, E, K, R, H)
● High entropy = low order - more H-bonding possibilities
Bases (K, R) - Acids (D, E)
a-Helices | Φ ≈ -65° Ψ ≈ -40° | Complete intra-backbone H-bonding | Van der waals core | hydrophobic face or hydrophilic face | amphipathic
The charge state of histidine changes at close to neutral pH
helices in transmembrane proteins
Native PAGE - Folded Protein
β-sheets | Φ ≈ -120° Ψ ≈ 120° | anti-parallel biased bc backbone connectivity | b-barrel common in enzymes | can be distant | aggregates =
SDS-PAGE - Unfolded Protein
pathology (AD, tauopathies, etc)
- Boiling - Unfold
Glycine and Proline don’t work. H-bonding directionality important.
- SDS - Unfold
- BME - Disulfide Bonds
● Fluorescence Quenching
● Circular Dichroism - Measures absorbance of circularly polarized light - difference in absorbance of left and right polarized light =
pKa = pH at which 50% of XH is dissociated into X- and H+
ellipticity | measures secondary structure | 222nm good because you can just look at neg/pos elip.
pI = the pH at which a molecule has no net charge
● UV-Raman Resonance
Ionic/Electrostatic/Hydrogen = Minor Stability Major Structure ● NMR
Hydrophobic/Van der waals = Major Stability Minor Structure
Purification
General Assays - UV Absorbance, Dye binding (Coomassie)
● UV Absorbance: Good for finding total protein, bad bc DNA contam and needs W or Y
Protein folding is entropically unfavored (it restricts motion and ● Dye Binding - Good for quantitative, assumes protein standard and unknown protein bind dye similarly,
decreases entropy) and therefore the TdeltaS contributes to a Specific Assays - functional properties
positive deltaG, and makes folding unfavorable. The hydrophobic
Fractionation
effect contributes to the DS(entropic) elements of protein folding. In
general, entropic contributions hurt the energetics of protein folding, - Organelle (Rate zonal (Sedimentation velocity), Eq. Density (Centrifugal force, buoyancy, and density)
but the hydrophobic effect increases entropy as proteins fold by - Solubility (Ammonium Sulfate precipitation/salting out - high salt concentrations favor hydrophobic interactions - proteins aggregate;
releasing more entropically free water molecules from the surface dialysis to remove the ammonium sulfate) (Isoelectric precipitation - salted out over narrow titration range)
of exposed hydrophobic residues. This offsets some of the entropic
- Size and Shape (Velocity sedimentation) (Gel-filtration “Sizing” - Can be used to estimate native molecular weight)
losses of protein folding

Plasmid System - Inducible T7 Promoter by LacR, His-tag,

Midterm 2 Kd
Why X-Rays? Wavelength. Weak Binding: KD = 10-4 - 10-5M.
Why Crystals? Increase signal but not noise. Averages of damages. Typical Binding: KD = 10-6 - 10-9M.
Protein might crystallize differently than in the cell. Very Strong: KD < 10-9
How does it work. Light interacts with matter. Incident beam is always stronger. The x–ray is very high energy and very little Binding Dynamics
diffracts. Dissociation is a first order reaction
To make a crystal, you purify then supersaturate by creating a ucleation site. We don’t know what makes proteins Dimerization is a second-order reaction
crystallize, but we add random things like PEG and MPD. Must prevent back reaction to accurately measure the rate constant
Fourier Transform - Wavelength. Amplitude. Phase. You need those two to calculate e- density. Pseudodimerization - Concentration of one reactant is so high that it does not significantly change during the reaction (virtuallyo constant) and
Phase Estimation. F(q) = |F(q)|eiΦ(q) can be included in an effective rate constant.
Experimental - Complete unknown. Surface plasmon resonance is a spectroscopic method for analyzing short-lived complexes. Measures Kon and Koff and therefore KD.
Molecular Replacement - Known - you get what you put in if you’re wrong. You replace structure with heavy electron dense Crowding = higher effective concentration. It can move the protein above the Kd for an interaction and stabilize binding.
to back-calculate what phases would be.
Refinement Resolution - High is 1 angrsontrg. Low is 3 angstroms.
Conformational issue, not side chain issue.
R/Rfree is a measure of bias.

Midterm 3
Meeting 17 - Introduction to Enzymatic Catalysis
Water acts as a nucleophile in solution. Peptide bonds are very stable despite negative free energy
of hydrolysis. Water is a bad nucleophile, but enzymes can orient the lone pairs towards the R-X.
You can add strong base to make water a better nucleophile. Proteins from dinosaurs still exist
because of low water environments which proves strong peptide bonds. ATP hydrolysis is very
favorable, because not spontaneous because the EA is very high. The highest energy state is the TS
and defined the rate of reaction. Law of kinetic significance says that significant reaction paths must
have a rate constant faster than the overall reaction.
Peptide bond hydrolysis in strong acid has good leaving group, and in strong base, has good
nucleophile. Cells can’t change pH, so they use histidine which is better in it’s deprotonated form.
Catalysts stabilize partial charges formed in transition states. The TS in a concerted reaction doesn’t
have the formation of partial charges. There are two mechanisms for catalysis of acyl compounds.
(1) Nucleophilic attack by covalent nucleophile (serine) (2) Concerted - Stabilizing
charges/Acid-Base. Serine and cysteine are the best amino acids that act as nucleophiles. Imidazole
can also act as both a base and acid (buffer).
Meeting 18 - Michaelis Menten
The two ways to make a reaction faster are by (1) lower energy of TS or (2) increase the energy of
substrate.
[S] >>> [E], so we ignore k-2. [E]t = [ES].
We can’t know [ES] except for at Vmax. KM is [S] where rxn rate = ½ Vmax.
k1[E][S] = k-1[ES] + k2[ES].
KM = (k2 + k-1)/k1
Lineweaver-Burke Plot plots 1/VObs over 1/[S] to linearize the plot. Specificity constant is KCat/KM. KCat
= K2.
Meeting 19 - Enzyme Kinetics/Inhibition
Avidity - A flexible linker can drive K1. Don’t need to pay as much entropic costs. Competitive
inhibitors change KM but not Vmax because 1000X [S] will outcompete inhibitor. K2 & K-1. IC50 is how
much inhibitor it takes to inhibit 50% of the reactions. Allosteric inhibitor can block k1 or k2. Can’t
outcompete allosteric inhibitor. KMax doesn’t have to change. Small molecule inhibitors can treat HIV
by binding reverse transcriptase allosterically.
Figuring out How Proteins are Doing Chemistry
Molecules that do hydrolysis are inhibited by DIFP. Covalent inhibitors mean that nucleophilic attack
by residues on the enzymes, not by H2O. They make the K-1 = 0.
I know the enzyme, but where is the active site?
Protein -> Mass Spec + TLC -> Serine -> Covalently modified by DIFP -> If I give the system a
nucleophile, it should restore function. -> ALso works with PAM which binds better an orients it’s -OH
towards the active site serine.
How is enzyme specificity conferred with identical active sites? The context. For example, ACHe has
a negative charge in it’s binding pocket that causes peptides to orient a certain way. NH4+ is a
competitive inhibitor. O-esters have better LG whereas N-esters are worse, but N-esters are native.
Meeting 20 - Defining a Reaction Path
Proteases have impressive KCat/KM given they catalyze reactions that otherwise wouldn't occur.
There are two models for hydrolysis by chymotrypsin. (1) Nuc attack and sequential release. (2)
Concerted hydrolysis. We can test by using a model substrate with an O-ester aromatic compound
and a radiolabeled ester. Sequential release of product does not conform w/MM. Protein folding is
slower than binding. Using model substrate allows us to trap covalent intermediates.
Meeting 21 - The Active Site
There are three R groups in the catalytic triad. Serine, Histidine, and Aspartate. OH on serine is bad nuc, so histidine accepts the proton. Aspartic acid can activate histidine by orienting it and stabilizing
positive charges. Aspartic acid increases the pKA of histidine and makes it a weaker acid. Proteolysis by chymotrypsin is pH dependent. Serine forms a covalent interaction with DIFP. TPCK binds
chymotrypsin whereas TLCK binds trypsin - although, they have the same active site but different KM, Catalytic triad = convergent evolution. You can mutate histidine in the catalytic triad and then add
substrate with histidine, and it rescues catalysis. To prove D102N didn’t cause protein unfolding, just add TLCK. Triple mutant still has 3-fold magnitude higher efficiency than uncatalyzed reaction
because of binding. At pH > 8.7, N-terminus becomes deprotonated. When it’s protonated, is forms salt bridge with Asp194 which enables binding. Covalent inhibitor can’t inhibit at high pH. Study with a
method whereby you titrate protons. Find that binding occurs on the ms timescale whereas folding is s. Use preincubated buffer experiment. Thermodynamic demonstrates induced fit. Three examples for
induced fit. Hexokinase, beef liver esterase, and chymotrypsin. Boltzmann distribution says that some amount of enzyme must already be active.
Meeting 22 - Enzyme Geometry + Metabolism
Metabolism is energy flow. ADP + Pi -> ATP. Glycolysis occurs in the cytoplasm, but sends pyruvate into the mitochondria, causing a sink which drives the reaction forward. Inputs are Glucose and ATP.
Outputs are 2 ATP, 2 Pyruvate, and 2 NADH. ΔG -34 because [ATP] in the cell is high. ΔG of ADP to ATP is 0.09 because ATP is high. Glucose has higher energy than ATP. All metabolic pathways are
connected. Regulation by isozymes - hexokinase has a lower KM & VMax. Citric acid cycle. Input is pyruvate. Output is 6 NADH and 2 FADH2. Enzyme symmetry is proven by forcing citrate into an
uncomfortable position. Radiolabeled experiment proves. NADH has higher energy than NAD+ due to more pi systems and resonance. Can create a TS analog and raise an antibody against it. Abzyme =
antibodies that do catalysis. Only one stereoisomer of the TS analog works which proves enzyme geometry matters. Good binders = poor enzymes. Lactate dehydrogenase makes the enzyme a good
binder and creates a sink to make the reaction favorable.
Meeting 23 - Cofactors
Iron is redox active. There are 20 amino acids, but only 1 is redox active. Proteins can do a lot, but they can’t do redox chemistry. Cofactors are engines. The electron transport chain contains a bunch of
cofactors like coenzymes, Fe-S clusters, Flavin Mononucleotide. They can lower pKa.
Can study cofactors using DNAse fotprinting
Meeting 24 - Coupled Vectorial Processes
Proton gradient explains everything.
Redox potential assesses the affinity of a compound for electrons. Teh higher the potential, the higher the affinity of the compound for electrons. CoQ needs a higher redox potential to grab electrons from
Fe-S cluster and move them to complex III.
Gamma-subunit uses the mechanical force generated by the proton gradient to induce a conformation change in the b-subunit of the ATPase and release ATP so more ADP + pi can bind, and ATP
synthesis can proceed.
What other explanation for 1c would fully explain the data while being consistent wiht the hypothesis of a concerted reaction in the enzyme’s active site?
If there is hydrolysis by water but different affinity for the two different products, the data would be indistinguishable from sequential product release.
Would doubling the substrate concentration affect the reaction? Use the reaction coordinate diagram to explain your answer.
Yes, doubling the substrate concentration will move this system out of equilibrium. Given that
the spontaneous reaction occurs in the direction of the dissociation, this would make this
reaction even more favorable. The starting free energy would be higher and thus the activation
energy would be lower. (Of course, as the mixture reacts towards equilibrium, the starting free
energy would decrease until it reaches the equilibrium level)
Can one change the rate-determining step in the enzymatic reaction by changing the
substrate concentrations? Briefly explain why or why not.
Yes! If the substrate concentrations are very low, the rate-determining step will be the formation
of the E…A-B complex; for high substrate concentrations, catalysis will be rate-determining.
Briefly explain why adding indole improves the ability of chymotrypsin to hydrolyze peptide
bonds after small residues (e.g. Ala, Gly, ...).
It could be because of an induced fit, for example: the binding energy of indole binding to the
specificity pocket is to some extent changing the active site into a structure that is more optimal
for peptide hydrolysis.
Why is it important to provide a large excess of substrate in this experiment?
Substrate concentrations need to remain roughly constant for the system to be in steady state.
(If the substrate concentration gets much lower over the course of the experiment, this will lead
to a change in the v obs over the course of the experiment and the system would not be in steady
state)
a) Briefly explain how catalysis can occur in the absence of the catalytic triad.
Enzymes work in part by stabilizing the transition state. The transition state will still be stabilized
even in the absence of the catalytic triad and this will facilitate the reaction.