BIOCHEM Exam 2

Includes lectures 5-11

What is the correct order of steps for protein purification?

1. Break open the cell 2. Centrifugation 3. Column chromatography 4. Gel electrophoresis 5. Analyze

What is ATCase regulation?

High amount of CTP product inhibits ATCase at

To study enzyme activity, structure, or sequence, a protein must be:

Completely isolated and purified

Purity of the isolated protein is analyzed by:

SDS-PAGE

Low-speed centrifugation removes:

Large debris, nuclei, cell walls

After centrifugation, the supernatant is:

The liquid containing soluble proteins

High-speed centrifugation separates:

Organelles (mitochondria, lysosomes)

Very high-speed centrifugation removes:

Ribosomes, membranes, small vesicles

The stationary phase is:

A solid material proteins stick to

The mobile phase is:

A liquid that carries proteins through the column

Chromatography separates molecules based on their:

Different interactions with the stationary phase

Column chromatography steps include:

Load column → add sample → elute → collect fractions

Size-exclusion chromatography separates proteins based on:

Size (molecular weight)

In gel filtration:

Large proteins elute first

Ion-exchange chromatography separates proteins based on:

Charge

The two main types of ion exchangers are:

Cation-exchange & anion-exchange

Proteins are eluted using:

Increasing amounts of competing ions (salt gradient)

SDS gives proteins a:

Negative uniform charge

SDS-PAGE gels are made from:

Polyacrylamide

In SDS-PAGE, proteins are separated based on:

Size

Negatively charged proteins in SDS-PAGE move toward:

Anode (+)

In SDS-PAGE:

Small proteins move fastest

Trypsin cleaves on the C-terminal side of:

Lys & Arg

Chymotrypsin cleaves on the C-terminal side of:

Trp, Tyr, Phe

Cyanogen Bromide cleaves proteins:

At methionine residues

Edman degradation determines:

N-terminal amino acid sequence

Edman degradation uses:

Phenylisothiocyanate (PITC)

Each Edman Degradation cycle removes:

The N-terminal amino acid

A catalyst increases reaction ________ without being ________.

speed; used up

Catalysis refers to the:

acceleration of a reaction

Without enzymes, most biochemical reactions would:

Occur too slowly to sustain life

ΔG° describes:

Free energy change from reactants → products

ΔG°‡ is the:

Activation energy barrier

The transition state represents:

The highest-energy intermediate

Enzymes work by:

Lowering ΔG‡

Enzymes:

Do NOT affect ΔG°

Glucose oxidation has ΔG° = –689 kcal/mol, meaning:

Reaction is spontaneous

A substrate is:

A reactant that binds the enzyme to form a complex

Active sites:

Contain unique amino acids specific for binding

Enzyme–substrate interactions are primarily:

Weak noncovalent interactions

The two major binding models are:

Induced-fit & lock-and-key

Lock-and-key model fails to explain:

Enzyme conformational change

Induced-fit is favored because:

It allows enzyme conformational change upon substrate binding

A perfect lock-and-key fit would:

Make the ES complex too stable

Once the substrate binds, the enzyme:

Converts ES → EP → E + P

To measure enzyme kinetics, you vary:

[substrate]

Km is:

[S] at ½ Vmax

Low Km means:

High enzyme affinity

Vmax is:

Maximum velocity when the enzyme is saturated

Lineweaver-Burk plots are useful because they:

Convert hyperbolic Michaelic-Menten curves into a straight line

On an Lineweaver-Burk plot:

y-intercept = 1/Vmax

Km is obtained from:

–1/x-intercept

A competitive inhibitor binds to:

Active site

Competitive inhibitors:

Increase Km, unchanged Vmax

Competitive inhibition can be overcome by:

adding more substrate

Noncompetitive inhibitors bind:

At a site other than active site

Noncompetitive inhibitors:

Lower Vmax, Km unchanged

In noncompetitive inhibition:

Substrate can still bind, but enzyme is inactive

Given that lines intersect at x-axis but have different y-intercepts, urea is:

Noncompetitive

Enzymes in pathways are regulated mainly to:

Save energy and avoid wasteful reactions

High-energy pathways must be regulated because they:

Are expensive in ATP

Stopping unnecessary steps in a pathway:

Reduces wasted intermediates

Which of the following is controlled by regulation?

Both

A zymogen is a(n):

Inactive enzyme precursor

Zymogens become active when they are:

Cleaved to change conformation

Cofactors are small molecules that:

Are required for enzyme activity

Which is a cofactor?

Both metal ions and coenzymes

Phosphorylation is the covalent addition of:

a phosphate group (PO₄³⁻)

Phosphorylation typically:

Causes conformational change and alters activity

Which residues can be phosphorylated?

Ser, Thr, Tyr

Kinases:

Add phosphates from ATP to proteins

Feedback inhibition occurs when:

Products of a pathway inhibit an earlier enzyme

Feedback inhibition:

Prevents waste and saves energy

A high concentration of final product means:

No need for more product → pathway should shut down

ATCase catalyzes a step in synthesis of:

CTP

CTP is primarily used for:

RNA and DNA synthesis

High CTP:

Inhibits ATCase

If CTP inhibits ATCase, the whole pathway:

Turns off to conserve energy

ATCase activity curve is:

Hyperbolic

The sigmoidal curve indicates:

Cooperative substrate binding

CTP causes ATCase activity to:

Decrease

ATP causes ATCase activity to:

Increase

ATCase is made of:

3 catalytic dimers + 2 regulatory trimers

CTP binds to the:

regulatory subunits

Binding of CTP causes:

Conformational change → decreased activity

ATP binding to ATCase:

Activates the enzyme by shifting to active conformation

ATP stimulates ATCase because:

Both ATP and CTP are required for nucleic acids; ATP signals energy availability

Phosphorylation typically changes:

Conformation and function of the protein

Phosphorylation can:

All of the above

Kinases transfer phosphate from:

ATP → protein

Dephosphorylation typically:

Removes PO₄ and causes a new conformation

Na⁺/K⁺ pump transports:

3 Na⁺ out, 2 K⁺ in

Phosphorylation causes the Na⁺/K⁺ pump to:

Dump Na⁺ to the outside of the cell

Dephosphorylation causes:

Conformational change returning pump to Na⁺-binding state

Biological membranes are primarily made of:

Lipids, proteins, carbohydrates

Membranes:

Serve as defining boundaries of cells and organelles

Membranes:

Also regulate molecular transport

Lipids are broadly defined as molecules that are:

Insoluble or poorly soluble in water but soluble in nonpolar solvents

Amphipathic lipids have:

Polar head + nonpolar hydrophobic tails

Sterols contain:

A four-ring structure