Midterm 1 Memorization + Enzyme Thermodynamics

What are the 3 methods for determining the structure of intact proteins?

1. cryo-electron microscopy: beam of electrons to visualize a frozen protein

2. nuclear magnetic resonance spectroscopy: measures the location of nuclei

3. x-ray crystallography: uses x-rays to measure electron density

list the 7 post translational modifications of proteins for bioc 202

1. phosphorylation

2. glycosylation 

3. hydroxylation 

4. carboxylation

5. acetylation

6. methylation

7. cleavage

what is phosphorylation and which amino acid residue(s) is it commonly associated with? 

• phosphate group attaches to -OH

• activates or inactivates a protein

associated with: (aa with -OH part of its R group)

• Serine, Threonine, Tyrosine

What is glycosylation and which amino acid residue(s) is it commonly associated with? 

• attachment of one or more sugars to a residue 

• purposes include surface labelling (most proteins on the cell surface are glycosylated) 

associated with: Asparagine, Threonine, Serine and Glutamine (all polar/hydrophillic) 

what is hydroxylation and which amino acid residue(s) is it commonly associated with? 

• addition of an OH (hydroxyl group)

• fibre stabilization: vital for collegen

• associated with: Proline

what is carboxylation and which amino acid residue(s) is it commonly associated with? 

addition of a carboxyl group

• usually to Glutamate

• clotting

what is acetylation and which amino acid residue(s) is it commonly associated with? 

addition of an acetyl group to an NH3+ (amino group)

• associated with Lysine

what is methylation and which amino acid residue(s) is it commonly associated with? 

the addition of a methyl group to electronegative atoms (N, O)

• associated with Arginine, Lysine, Glutamate, Aspartate

what is cleavage and which amino acid residue(s) is it commonly associated with? 

most proteins are trimmed after synthesis to activate or inactivate them

• multiple proteins from one polypeptide is common 

enzyme

biological macromolecule (large molecule; carbohydrates, lipids, proteins, nucleic acids), usually a protein, that acts as a catalyst for biological reactions

• highly specific and only catalyze a particular set of reactions 

catalyst

a substance that increases the rate of a chemical reaction without itself undergoing any permanent change

• they do this by lowering activation energy 

kinetics 

the branch of chemistry concerned with the rates of chemical reactions

• enzyme kinetics studies how enzymes bind to their substrates and turn them into products 

substrate

a molecule that an enzyme acts on

• the substrate binds to the enzyme’s active site through a series of weak interactions 

apoenzyme

an enzyme that is inactive because it is missing its cofactor (a required non-protein component) 

cofactor

an inorganic ion or a small organic compound required for an enzyme’s activity

holoenzyme

a complete, catalytically active enzyme, consisting of an apoenzyme combined with its necessary cofactor(s)

prosthetic group 

a cofactor that is tightly or covalently bound to the active site of an enzyme. A heme group is a common example 

transition state

(X‡)

a temporary high energy and unstable state that a substrate must pass through to be converted into a product. It is the point of highest free energy in the chemical reaction (because old chemical bonds are in the process of breaking and new ones are just beginning to form) 

• also has the lowest concentration

how exactly does an enzyme catalysis work?

• the active site of an enzyme acts as a perfect tool for the high-energy unstable “in-between” molecule that exists at the transition state

• the enzyme’s active site fits the transition state in two critical ways: 

  • shape: the 3D shape of the active site pocket is precisely arranged to fit the specific bond angles and shape of the transition state molecule

  • charge: the arrangement of amino acids within the active site creates a specific chemical environment to stabilize it 

*by being perfectly suited to hold and stabilize this unstable transition state, the enzyme drastically reduces the amount of energy required to form it, therby reducing the activation energy and speeding up the reaction

activation energy

(ΔG‡)

the minimum amount of energy required for reactants to transform into the transition state

• enzymes speed up reactions by lowering this energy barrier 

binding energy

(ΔGʙ)

• the energy released when an enzyme binds to and stabilizes the transition state

• this energy is the primary source for lowering the activation energy 

rate of reaction (V, velocity)

The speed at which a reaction proceeds, typically measured as the concentration of product formed per unit of time. Scientists use the initial velocity because at this point, the substrate concentration is at its highest and there is virtually no product yet, which simplifies the measurement and prevents other factors (like the reverse reaction) from influencing the rate.

Rate Constant (k) 

a proportionality constant that relates the rate of a reaction to the concentration of the reactants 

V0

initial velocity

• the reaction rate measured at the beginning of the reaction, when the concentration of the product is negligible 

Michaelis-Menten Equation

What It Is: The Michaelis-Menten equation is a mathematical formula that describes the speed (velocity) of a reaction catalyzed by an enzyme. It connects the initial reaction rate (V₀) to the concentration of the substrate ([S]). This allows us to predict how fast an enzyme will work under different conditions.

Where:

• V₀ is the initial rate of the reaction.

• Vₘₐₓ is the maximum possible rate when the enzyme is completely saturated with substrate.

• [S] is the concentration of the substrate.

• Kₘ is the Michaelis constant, representing the substrate concentration at which the reaction rate is half of Vₘₐₓ.

Key Shortcut: A crucial concept for solving problems quickly is the definition of Kₘ. If you are told that the reaction is proceeding at exactly half of its maximum speed (V₀ = ½Vₘₐₓ), you immediately know that the substrate concentration must be equal to the Kₘ value ([S] = Kₘ).

Michaelis Constant (Km)

The substrate concentration required for the enzyme to reach half of its maximum velocity (½ Vₘₐₓ). The Kₘ value is a crucial indicator of an enzyme's affinity for its substrate.

• Low Kₘ: Indicates high affinity. The enzyme can work efficiently even at low substrate concentrations.

• High Kₘ: Indicates low affinity. The enzyme needs a lot of substrate to be present to work efficiently.

Vmax 

The absolute fastest rate at which an enzyme can catalyze a reaction. This speed limit is reached when the enzyme is fully saturated with substrate—meaning every single active site on every enzyme molecule is occupied and working. Adding more substrate at this point will not make the reaction go any faster.

kcat (turnover number)

The maximum number of substrate molecules a single enzyme active site can convert into product per unit of time. It is the most direct measure of an enzyme's catalytic efficiency and represents its intrinsic top speed when fully saturated.

irreversible inhibition

occurs when an inhibitor binds to an enzyme, often covalently and permantely deactivates it.

• aspirin for example, irreversibly inhibits the cyclooxyrgenase (COX) enzyme 

reversible inhibition

occurs when an inhibitor binds non-covalently to an enzyme, and the enzyme-inhibitor complex can dissociate 

• this type of inhibition can be overcome 

competitive inhibition

a type of reversible inhibition where the inhibitor resembles the substrate and competes for the same active site.

non-competitive inhibition

a type of reversible inhibition where the inhibitor binds to a different site on the enzyme (an allosteric site), changing the enzyme's shape and reducing its activity.

• alanine for example acts as a non-competitive inhibitor for the enyme pyruvate kinase

lineweaver-burk plot

a graphical representation of enzyme kinetics, obtained by plotting the reciprocal of the initial velocity (1/V0) against the reciprocal of the substrate concentration. It linearizes the michaelis-menten equation making it easier to determin Vmax and Km 

what are the 3 examples of enzymes introduced and what do they do?

1. papain: speeds up the cleaving of any peptide bond

2. trypsin: speeds up the cleaving of only the peptide bond on the carboxyl end of Lysine and Arginine

3. thrombin: speeds up the cleaving of only Arginine-Glycine peptide bonds

characteristics of the active site

• they are clefts (aka dimples on the enzyme made up of residues from all over the active site) 

• occupy a small volume of the enzyme 

• water is excluded or manipulated from the active site 

• the substrate is bound to the active site by a series of weak interactions 

what is required for an enzyme to function?

it must be partly complementary to its substrate

• the general 3D shape of the substrate must match the shape of the active site and the chemical properties (pos, neg, oily, watery region) of the substrate must align with the corresponding properties in the active site 

BUT not perfectly…

• the substrate and enzyme need a good enough initial fit to bind to each other but the initial binding causes the enzyme to slightly change. its shape, clamping down or wrapping around the substrate more tightly 

• this creates a more precise and stable connection, perfectly positioning the substrate for the chemical reaction to occur 

explain and connect the lock and key and induced binding models 

• The Lock and Key model is the original, simpler idea. It proposes that the enzyme's active site is a rigid structure that is already a perfect geometric and chemical match for its specific substrate, just like a key fits into its one specific lock.

• The Induced Fit model is the modern and more accurate refinement. It states that the enzyme's active site is flexible, not rigid. The initial binding of the substrate is good but not perfect, and this interaction induces the active site to change its shape to wrap more tightly around the substrate.

• The connection is that the Induced Fit model builds upon and improves the Lock and Key concept. It keeps the essential idea of a specific match but adds the crucial element of enzyme flexibility. This flexibility is what allows the enzyme to stabilize the reaction's transition state, which is the key to speeding up the reaction.

do enzymes alter the final equilibrium of products to reactants or alter the change of free energy of the reaction?

no

can enzymes change the spontaneity of a reaction, for example make a non-spontaneous reaction with positive change in gibbs free energy spontaneous? 

no 

is the active site of the enzyme perfectly complementary to the transition state?

yes 

what do the following symbols represent? GS, GP, GX‡

• GS​ is the starting energy level of the Substrate.

• GP​ is the final energy level of the Product.

• GX‡​ is the energy of the Transition State—the highest-energy, most unstable point in the middle of the reaction.

what is the formula for the activation energy for the forward reaction

(the amount of energy needed to get from the starting energy level of the substrate up to the peak of the energy hill)

what is the formula for the activation energy for the reverse reaction

(the amount of energy required to get the energy level of the product back up to the same energy hill) 

reaction pathway (physical steps of an enzyme reaction)

formula for binding energy

(the total reduction in activation energy is equal to the binding energy that the enzyme provides by perfectly gripping the transition state)

what bond joins amino acids

peptide bonds

what is phi and psi angles?

phi: dihedral angles of N-Ca

psi: dihedral angles Ca-C=O

what are the characteristics of the bonds in a polypeptide chain 

1. C=O to NH bond is locked due to double bond character as a result of resonance; results in same plane

2. psi and phi bonds (other bonds beside 1) have free rotation 

3. BUT not all combinations of psi and phi angles are possible (combinations defined by ramachandran plot)

what are the psi and phi angles for alpha helices 

phi = -60 degrees 

psi = -45 degrees

(minimizes steric hinderance and allows for H-bonding)

what is the intrachain H bonding pattern for alpha helices

i (C=O) binds with i + 4 (N-H)

• all C=O and N-H bind except for the ends 

how are the R groups arranged in alpha helices

all perpendicular the backbone (R groups stick out)

how is the helix rise affected per amino acid residue added

1.5 A (angstrom) per aa residue

what is the pattern for R groups that face the same direction (interact with the same molec) for alpha helices 

i, i + 3, i + 4 

• this is because R groups are about 100 degrees apart (a full round is 360 degrees)

• i (0), i + 3 (300), i + 4 (400)

what are psi and phi for anti-parallel beta sheets

phi: -139

psi: +135

what are phi and psi for parallel beta sheets

phi: - 119

psi: +113 

what is the H bonding pattern for antiparallel beta sheets

linear

i -- j

C=O on aa i bonds with N-H on aa j 

what is the H bonding pattern for parallel beta sheets 

C = O i bonds with N-H j + 2 

N - H i bonds with C = O j 

• less stable, angled bonding 

how is the length of antiparallel and parallel beta sheets affected by the addition of an amino acid residue 

antiparallel: + 3.5 A per aa residue 

parallel: + 3.25 A per aa residue 

psi and phi dihedral angles for tertiary structure

aligns with ramachandran plot

acidic aa + R groups 

1. aspartate: 1 c, carboxyl 

2. glutamate: 2c carboxyl 

basic aa + R groups

1. histidine: 1 c, imidazole (5 mem, N and NH) 

2. arginine: 3c, guanidinium

3. lysine: 4c, amino 

special aa + side chains

1. cysteine: 1c, thiol (disulfide bonding)

2. selenocysteine: 1c, SeH 

3. proline: 5 mem w/ amino (NP), R config 

4. Glycine: NONE (NP) (achiral) 

polar aa + R groups

1. Asparagine: 1 c, amide 

2. Glutamine: 2 c, amide 

3. Threonine: 2 c, OH branch 

4. Serine: 1 c , OH

hydrophobic (mostly np) aa + R groups

1. Alanine: 1 c

2. Valine: 2 c, 1 c branch 

3. Isoleucine: 3 c, 1 c branch (1) 

4. Leucine: 3 c, 1 c branch (2) 

5. Methionine: 2 c, S, 1 c

6. Phenylalamine: 1 c, phenyl 

7. Tyrosine: 1 c, phenyl, OH (opp starting c)

8. Tryptophan: 1 c, indole (5 mem NH (3), 6 mem)

which aa have R groups with no carbon chains

glycine and proline 

which aa have R groups with 1 carbon chains

cys (2), rings (4), asp (2), alanine + serine

• cysteine

• selenocysteine

• phenylalamine

• tyrosine

• tryptophan

• histidine

• asparagine

• aspartate

• alanine

• serine

which aa have R groups with 2 carbon chains 

Glu, MVP

1. Glutamine 

2. Glutamate 

3. Methionine 

4. Valine 

5. Threonine (Polar) 

which aa have R groups with 3 carbon chains

Leu (2), Arg

1. Leucine 

2. Isoleucine 

3. Arginine 

which aa has a R group with a 4 carbon chain

Lysine

What are the amino acids for the following 1 letter codes:

R, N, D, Q, E, K, U, Y, W, F, T, A 

Arginine, Asparagine, Aspartate, Glutamine, Glutamate, Lysine, Selenocysteine, Tyrosine, Tryptophan, Phenylalamine, Threonine, Alanine

what are the 3 letter codes for the following amino acids : 

Glutamate, Aspartate, Glutamine, Asparagine, Isoleucine, Tryptophan, Selenocysteine 

Glu, Asp, Gln, Asn, Ile, Trp, Sec

what is the exception to favouring trans peptide bonds

X-Proline

• some can be cis bc both have steric hinderance (the energy diff between cis and trans is smaller compared to the diff w other aa) 

coulomb’s constant

proportionality constant (depends on dielectric constant); converts to the correct units to end up with Newtons

coulomb’s law

pKa

specific pH at which a functional group is exactly 50% protonated and 50% deprotonated (tipping point)

• buffering zone is when pKa is within ± 1 of the pH (a certain amount of acid/base needs to be added to overcome this 

what are the functional groups among the amino acids

hydrophobic 

• alkyl (just carbon; alanine, valine, isoleucine, leucine)

• phenyl (phenylalamine) 

• phenol (tyrosine) 

• indole (tryptophan; 5 mem ring, NH (3) + 6 mem) 

• thio-ether (methionine: c - s - c)

• pyrrolidine (proline) 

hydrophilic 

• hydroxyl (serine, threonine) 

• amide (asparagine, glutamine) 

• thiol (cysteine; can form disulfide bonds, weakly H bonds)

• SeH

basic (neutral to +)

• imidazole (histidine; 5 mem, N (2), NH (3) 

• guanidinium (arginine; NH, NH, NH2)

• side-chain amino (lysine) 

acidic (neutral to -)

• side chain carboxyl (aspartate, glutamate) 

which aa has constrained phi rotation?

proline 

strongest interaction among aa 

cysteine - cysteine 

• disulfide bond (covalent) 

a-helix wreckers

proline + glycine