Lecture 4.1

Question 1

What are ligands?

Ligands range from:

Ligand binding occurs at the:

How?

Answer 1

bound molecules (reversible/non-covalent)

range from small molecules to other proteins

ligand binding occurs at the binding site. binding sites are complementary to ligand (H bonds, ionic interactions, hydrophobic interactions, van der Waals interactions, shapes/sizes, charge)

Question 2

When we graph binding, we usually plot x axis= and y axis=

Answer 2

x axis = ligand concentration

y axis = fraction of protein bound (or activity)

The shape of the curve tells us how binding behaves

Question 3

What is non-cooperative binding?

Answer 3

happens when a protein has one binding site or multiple sites that don’t influence each other

curve looks like a smooth hyperbola (like Michaelis Menten)

means that each ligand binds independently

binding one molecule does not change how easily the next one binds

i.e. myoglobin binding oxygen

(gradually saturates as concentration increases)

Question 4

What is cooperative binding?

Answer 4

Happens when the protein has multiple binding sites, so binding at one site changes the shape of the protein which affects other sites

S-shaped, sigmoidal curve = allostery

at low ligand concentration, binding is harder. once one ligand binds, the protein changes shape, and other sites bind ligand more easily

i.e. hemoglobin binding oxygen

NOTE; sigmoidal = allostery = binding at one site affects another site

cooperativity is a type of allostery

positive cooperativity: binding makes more binding easier

negative cooperativity: binding makes further binding harder

Question 5

What is the ligand binding equation that gives the hyperbolic binding curve?

Answer 5

Start with the binding reaction
P+L⇌PL
P = free protein, L = free ligand, PL = protein-ligand complex

Define the fraction of protein bound, theta
θ=[PL]​/[P]+[PL]
meaning, fraction bound = bound protein / total protein

Introduce the dissociation constant
Kd​=[PL]/[P][L]​

Meaning: low Kd = tight binding
High Kd = weak binding

Rearrange to isolate [P]
[P]=Kd​[PL]​/[L]

Replace [P] in θ=[PL]​/[P]+[PL]

θ=[PL]​ / ((Kd​[PL]​/[L]) + [PL])

To get

θ=[L]​ / Kd​+[L]

Which describes simple ligand binding

Graphically, if you plot x-axis = [L] and y axis = fraction bound θ, you get a hyperbolic curve

Question 6

In a hyperbolic curve equation, when [L] = Kd​

Answer 6

θ = 0.5

Note Kd is equilibrium constant

θ is fraction bound

Question 7

Smaller Kd means

Answer 7

High affinity

Question 8

Enzyme active sites are analogous to a ligand binding site except

Answer 8

it also catalyzes a reaction

Question 9

What are cofactors?

Answer 9

Additional chemical components that assist in enzyme function

Can be metal ions, coenzymes, prosthetic groups, metal ions or coenzymes that are tightly or covalently associated with a protein

Question 10

What is a holoenzyme/holoprotein? Apoenzyme/apoprotein?

Answer 10

holoenzyme: enzyme (protein) + prosthetic group

apoenzyme: enzyme (protein) without prosthetic group

Question 11

Enzymes reduce what of a reaction?

Answer 11

activation energy (∆G‡)

Note that ∆∆G‡cat is the efficiency of the catalyst

Question 12

Multistep reactions. Reaction mechanisms may involve multiple:

Overall rate is determined by:

Answer 12

transition states and intermediates

Rate limiting steps will determine the overall rate

Question 13

For activation energy, the rate of a reaction is related to the

First order reaction equation:

Lowering ∆G‡ exponentially increases what

Answer 13

concentration of substrate and a rate constant (k)

V = k[S]

increases the rate constant k

Question 14

a catalyst increases the rate of carbonic anhydrase reaction by how much?

Answer 14

10^7 fold rate enhancement

Question 15

Enzymes are classified by

Answer 15

reactions and processes they catalyze

class 1 = oxidoreductases
class 2 = transferases
class 3 = hydrolases
class 4 = lyases
class 5 = isomerases
class 6 = ligases
class 7 = translocases

Question 16

Describe class 1 enzymes

Answer 16

oxidoreductases

catalyze oxidation-reduction (redox) reactions

in this reaction
electrons or hydrogens are transferred between molecules

naming rule: classified as donor:acceptor oxidoreductase
donor = molecule being oxidized (loses electrons). acceptor = molecule being reduced (gains electrons)

enzyme types in this class
- dehydrogenases = remove hydrogen from substrates
- oxidases = transfer electrons to oxygen
- reductases = catalyze reductions

Question 17

Describe class 2 enzymes

Answer 17

transferases

catalyze the transfer of a functional group from one molecule to another

instead of moving electrons, they move chemical groups

naming rule:
donor: acceptor grouptransferase
donor = molecule giving the group
acceptor = molecule receiving the group

common groups transferred: phosphate, methyl, glycosyl, acyl

important subgroup: kinases: transfer phosphate groups, usually from ATP

example
ATP+protein(Ser)→ADP+protein(phospho-Ser)

Question 18

What is the more common name of the enzyme
ATP:D-hexose 6-phosphotranferase (EC 2.7.1.1)?
A. Phosphoglucomutase
B. Phosphofructokinase-1
C. Phosphofructokinase-2
D. Hexokinase
E. Pyruvate Kinase

Answer 18

D hexokinase

Question 19

Describe class 3 enzymes

Answer 19

hydrolases

catalyze hydrolyzes reactions (breaking a bond using water)

Substrate + H2​O → Product 1 + Product 2

Water splits into H (goes to one fragment) and OH (goes to other fragment)

Common hydrolases: proteases, lipases, nucleases, collagenase, hyaluronidases

Question 20

Describe class 4 enzymes

Answer 20

lyases

break bonds without water and without redox reactions

They break C-C, C-O, C-N bonds, do NOT use hydrolysis or involve oxidation

Often remove a small molecule (like CO2 or H2O), create a double bond, or form a ring

Single substrate→Product+small molecule

Or in reverse:

Two substrates → One product
Two substrates→One product

**Two substrates in one direction, one fewer compound in the other direction

Examples
Decarboxylases remove CO2
Dehydratases remove H2O
Often called synthases

Question 21

Describe class 6 enzymes

Answer 21

ligases

ligases join two molecules together, requires energy from ATP

General reaction

A+B+ATP→A−B+ADP+Pi
​
or

A+B+ATP→A−B+AMP+PPi

ATP is broken to drive the bond formation

often called synthetases (i.e. DNA ligase, glutamine synthetase)

key idea: bond formation, ATP req
​

Question 22

Describe class 5 enzymes

Answer 22

Isomerases

catalyze structural arrangements within the same molecule

no atoms are added or removed - the molecule is just rearranged

A→A′

same formula, different structure

example

Glucose-6-phosphate → Fructose-6-phosphate

This happens in glycolysis.

Both molecules have the same atoms but the carbonyl moves.

Common names: isomerase, mutase, racemase, epimerase

i.e. phosphoglycoisomerase

Question 23

Describe class 7 enzymes

Answer 23

translocases

move molecules across membranes

example: moving ions across a membrane, transporting molecules into or out of cells

i.e.
Na⁺/K⁺ ATPase
Moves:
Na⁺ out
K⁺ in

Uses ATP to power transport.

key idea: membrane transport, often uses ATP or ion gradients

Question 24

Overview of 7 enzyme classes

Answer 24

1) oxidoreductases: redox reactions

2) transferases: transfer functional groups

3) hydrolases: break bonds using water

4) lyases: break/form bonds without water or redox

5) isomerases: rearrange atoms within molecule

6) ligases: join molecules using ATP

7) translocases: move molecules across membrane

Question 25

the EC (enzyme commission) number is a 4 number classification system that tells you exactly what type of reaction an enzyme catalyzes. What does EC 3.4.21.4

Answer 25

1st number: enzyme class (type of reaction) (3 = hydrolase) 2nd number: substrate type (4 = acting on peptide bonds (this hydrolase cuts proteins, meaning its a protease/proteinase)) 3rd number: mechanism or catalytic type (21 = serine protease, tells us how the enzyme works mechanistically) 4th number: specific enzyme within that subgroup (4 = chymotrypsin)