Basic Biochemistry

Question 1

A cell is tasked with creating order (structure) from disorder (raw materials). What does this require?

Answer 1

Energy, raw materials that you can break and can build up, instructions, enzymes to drive chemical processes

Question 2

The chemical processes by which the cell drives its function.

Answer 2

Metabolism

Question 3

Breaking down to make raw materials and energy (oxidizing)

Answer 3

Catabolism

Question 4

Building up macromolecules with energy

Answer 4

Anabolism

Question 5

Despite the creation of order, we’re not violating the 2nd Law of Thermodynamics

Answer 5

Order is paid for by the discharge of heat into the enviornment
-fun fact: we burn off 1000-1500 kcal by just being human

Question 6

All reactions have a change in free energy (delta G)

Answer 6

• delta G takes entropy, enthalpy, and temp into account

- Can only tell us what can be done– not if and how fast!

Question 7

(-) delta G

Answer 7

spontaneous reaction

-rxn favors product and has some small degree of substrate (at equilibrium implies more product than substrate)

Question 8

(+) delta G

Answer 8

nonspontaneous reaction

-can undergo a reaction if coupled to a spontaneous rxn.

Question 9

Some cellular problems of reactions (even if rxn is spontaneous)

Answer 9

• Rxn can occur, but won’t w/out some sort of kick start
• Rxn can and does occur, but not fast enough
• Rxn can occur, but substrate(s) are not in the correct place or time

Question 10

What is needed for a rxn to take place in a cell?

Answer 10

Enzymes! To overcome cellular problems of rxns. we need an enzyme– a biological catalyst– and the proper compartmentation.

Question 11

Activation energy: Minimum of energy needed in a “collision” between 2 molecules that will result in a rxn.

Answer 11

• Enzymes lower the amount of bombardment needed to get a rxn.
• Random collisions can get you near the peak of Ea
• Free energy (delta G) isn’t changed!

Question 12

In cells, not many molecules can reach the Ea alone. Why is this useful?

Answer 12

Control!

Question 13

Reactants that are below Ea, will not react unless something pushes them to the Ea

Answer 13

Metastable state

Question 14

How do we control enzymes in the cell?

Answer 14

• feedback inhibition from downstream products
• physical separation of enzymes and substrates (compartmentalization)
• can use other chemicals
• gene promoters can control txn
• control mRNA and protein degredation

Question 15

Speeds up rxn rate but isn’t changed in the end.

Answer 15

Catalyst

Question 16

Basic properties of BIOLOGICAL CATALYSTS (enzymes)

Answer 16

1. increases the rate (probability) of rxn by lowering Ea: rxn occurs w/out thermal activation
2. Forms a transient interaction w/ the substrate(s), facilitating interaction
3. Changes only the rate: cannot make a (+) delta G move forward w/out added energy. Rate can increase greatly
4. No changed during the rxn
5. Can be proteins (most are and end in -ase) or RNA
6. Most are highly specific
7. Readily coupled to other enzymatic rxns.

Question 17

How do you couple?

Answer 17

Harness energy by linking molecules together. Couple favorable with unfavorable.

Question 18

Substrate binding

Answer 18

“lock and key”

• “induced fit” is what we really call it
• maximizing chemical rxns between substrate and enzyme
• shape will bend and twist to get the induced fit
• when enzyme and substrate are in complex with each other, that’s the Ea hump.

Question 19

Features of Enzymes

Answer 19

Some have a tightly bound prosthetic group: metal ion, heme, etc…, for function (often electron acceptor)
-Folding dictates active site

Question 20

A pocket or groove formed by amino acids (tertiary structure) where the substrate(s) bind.

Answer 20

Active Site

Question 21

Enzymes can be hyper-specific: succinate dehydrogenase

Answer 21

• Name refers to the reversible process
• Catalyzes the reduction of fumarate into succinate
• Enzymes are named for only one direction of the rxn, so it can be misleading

Question 22

Types of enzymes to memorize!

Answer 22

pg 144, table 4-1

Question 23

Redox rxns “LEO GER”

• To tell if redox rxn, look for:
  1. charge differences
  2. oxygen
  3. protons
  4. formation/loss of double bonds

Answer 23

Oxidoreductases

Question 24

Transfer of functional groups from one molecule to another (kinases: cause phosphorylation)

Answer 24

Transferases

Question 25

Hydrolytic cleavage of a molecule (nucleases, proteases, phosphatases, etc...) - Using H2O to break stuff - condensation is the reverse process

Answer 25

Hydrolases

Question 26

Movement of a functional group within a molecule

Answer 26

Isomerases

Question 27

Joining 2 molecules together (polymerases, ligases--often are condensation rxns)

Answer 27

Synthases

Question 28

Joining of 2 molecules or breaking 1 into 2 with an electron rearrangement (break or form double bonds) - When breaking apart, one molecule will get a double bond - When join together, a molecule will lose a double bond

Answer 28

Lyases

Question 29

Conditions affecting enzymes

Answer 29

Temp | pH

Question 30

T or F: enzymes do not remain static.

Answer 30

True

Question 31

How do we get the enzyme and substrate together?

Answer 31

All has to do with rate: - diffusion - scaffolding: put them in some assembly together - limiting space of diffusion (ex vesicles)- still diffuse, but space is limited - activation of proximal proteins- can put them next to each other, but can't react with some chemical change in the environment happens.

Question 32

Enzyme kinetics

Answer 32

when rate of rxn and substrate concentration graph starts to level off, all enzymes are occupied by substrate. -called Vmax, and can be increased if enzyme amount is increased LOOK AT NOTES FOR EXTRA INFO

Question 33

Concentration needed to reach 1/2 max V.

Answer 33

Km

Question 34

Michaelis-Menten equation

Answer 34

V= Vmax [S] / Km + [S]

Question 35

When [S]

Answer 35

then v=Vmax [S]/Km , and initial reaction velocity is roughly proportional to [S]: 1st order region f the M-M plot

Question 36

When [S] > Km,

Answer 36

Then V=Vmax[S]/[S] or V-Vmax, or 0 order rxn where substrate concentration no longer matters

Question 37

What dictates Vmax?

Answer 37

Enzyme concentration

Question 38

When [S]=Km, then

Answer 38

V=Vmax/2. Thus, Km is the point at half Vmax: this is the Michaelis constant (Km)

Question 39

Enzyme regulation: how else can we control enzyme reactions? Why would this be important?

Answer 39

Feedback inhibition implies that product comes back to inform of a change. - Enzyme can be informed b/c of its multiple binding sites - downstream or immediate products can bind to regulate by changing enzyme shape.

Question 40

Inhibition of Enzymes

Answer 40

Competitive inhibition | Noncompetitve inhibition

Question 41

Inhibitor and substrate both bind to the active site of the enzyme. Binding of an inhibitor prevents substrate binding, thereby inhibiting enzyme activity -inhibitor looks like substrate

Answer 41

Competitive inhibition

Question 42

Inhibitor and substrate bind to different sites. Binding of the inhibitor distorts the enzyme, thereby decreasing likelihood of substrate binding. - Inhibitor binds to somewhere other than active site - Also called Allosteric

Answer 42

Noncompetitive inhibition

Question 43

Regulation of an enzyme with a molecule that is not the substrate or product- binds to another site. -Often at the level of a "committed step" which will possibly have activated and inhibitory sites

Answer 43

Allosteric regulation

Question 44

Allosteric regulation

Answer 44

Allosteric inhibition | Allosteric activation

Question 45

An enzyme subject to allosteric _________ is active in the uncomplexed form, which has a high affinity for its substrate. When the allosteric _______ binds, the enzyme is stabilized in its low affinity form, resulting in little or no activity.

Answer 45

Allosteric Inhibition

Question 46

When the allosteric ________ binds to the enzyme, the enzyme turns into its high affinity form, resulting in enzyme activity

Answer 46

Allosteric activation

Question 47

Types of inhibitors (Chemical modifications)

Answer 47

1. Irreversible | 2. Reversible

Question 48

Covalently bound inhibitors (nerve gases, penicillin, asprin) that can be done in cells as well---phosphorylation -refers to not being able to spontaneously be removed

Answer 48

Irreversible inhibitors

Question 49

Inhibitors that can bind and dissociate (can be competitive or noncompetitive); noncovalent bonds

Answer 49

Reversible inhibitors

Question 50

Basic protein functions | *note: none of these work without proper structure--protein folding!

Answer 50

- enzymes - motor proteins - receptors - structural proteins - storage - gene regulation - transport - signaling - can have overlap of functions too

Question 51

Proteins form by:

Answer 51

polymerization of amino acids

Question 52

Protein folding happens by

Answer 52

Chemical interactions between side chains or backbone. Also covalent and hydrophobic interactions can dictate folding. -folding creates binding sites and specificity

Question 53

Levels of protein organization

Answer 53

1, 2, 3, 4

Question 54

Composition of amino acids in a polypeptide chain

Answer 54

primary protein structure

Question 55

structures formed by the backbone: alpha-helicies and beta-sheets; localized backbone interactions; atoms H bond with themselves

Answer 55

Secondary protein structure

Question 56

3D; formed by interactions of R groups of a single polypeptide chain

Answer 56

Tertiary protein structure

Question 57

Multiple polypeptide chain interactions; also 3D

Answer 57

Quaternary protein structure

Question 58

Active site is made possible by

Answer 58

folding of R groups

Question 59

What happens with a substrate is bound?

Answer 59

- substrate binds weakly to R group components - distortion of bonds-opening up to catalytic attack (hight energy state; activation energy) - enzyme accepts or donates proton, changing the reactivity of a substrate (pH sensitive) - accepts or donates electrons, formation of temporary (high energy) covalent bonds - stablilzes "unstable" bonds by all favorable rxns it makes with substrate

Question 60

Analysis of proteins: electrophoresis after breaking cells open

Answer 60

- unfolded tertiary structure - need to uniformly charge proteins by adding a detergent to ensure negatively charged proteins that will not escape gel.

Question 61

What do you need for gel electrophoresis?

Answer 61

- Gel matrix: polyacrylamide - Buffer - Denaturing agents: to make consistent band lengths (heat) - Anionic detergent (SDS): to give uniform charge, thus uniform separation

Question 62

SDS-PAGE

Answer 62

Sodium dodecyl sulfate polyacrylamide gel electrophoresis

Question 63

Things to do after SDS-PAGE

Answer 63

- stain and visulaize the sizes and amounts of proteins present - bring out of 3D into 2D - transfer proteins to a membrane and probe for specific proteins - use antibodies (Western blot) or molecules that bind to other proteins (secondary Ab) - elute proteins off gel and ID via mass spec. - enzymatic rxns in the gel

Question 64

Problems with transferring proteins to a membrane and probing for specific proteins after SDS-PAGE

Answer 64

-protein is denatured, so epitopes may be no more

Question 65

Immunoprecipitation

Answer 65

- break open cells - add and Ab to bind to a specific protein in the cell extract - add an agarose bead that contains a protein that will bind the Ab you added - Spin down the beads and wash away the rest of the non-Ab bound extract - remove the Ab/protein complex from the beads - add the soln that was precipitated by the Ab to an SDS-PAGE - Western blot for proteins that have been precipitated