BIOCHEM

What are enzymes?

Proteins that speed up chemical reactions *without being consumed or changed*

What is the difference between an apoenzyme and a holoenzyme?

APOENZYME:
inactive enzyme, activation occurs upon binding of an organic or inorganic cofactor

HOLOENZYME:
apoenzyme together with its cofactor. A complete and catalytically active enzyme

Enzymes are \____________ proteins and can sometimes be \_____ molecules

globular, RNA

Enzyme structure is determined and stabilized *mainly* by which force?

hydrophobic interactions

What properties do enzymes have that non-biological catalysts do not?

- higher reaction rates
- milder reaction conditions
- greater reaction specificity
- *capacity for regulation*

What allows regulation in enzymes?

confirmational changes in protein structure

Why is ATP a "high energy" molecule?

1. products have decreased electrostatic repulsion making product more stable than bond
2. increased resonance stabilization of inorganic phosphate
3. increase in entropy

All together these factors cause the release of \>-25kJ/mol of energy when broken

In a *biological system* when will a reaction proceed with respect to its free energy

only thermodynamically favorable reactions where free energy is negative, exergonic (products have less G than reactants)

Which state has the highest free energy?

Transition state

How can speed of a reaction be determined?

size of the activation energy barrier
--\> greater the activation energy the slower the reaction

What enzymatic factors lower activation energy?

1. desolvation - removing substrates from aqueous solution (don't have to compete for bonding)
2. Proximity and orientation
3. Taking part in reaction mechanism
4. stabilizing transition state!!!!
(binding site is most similar to TS)

Lock and key model of enzyme action

a now-discarded theory that enzymes are rigid and precisely shaped to fit only certain substrate molecules

induced fit model of enzyme action

the current theory, which states that enzymes are flexible, bend to fit the substrate

What are acid-base catalysts

give and take protons

\-- take part of reaction mechanism --

What are cofactors?

non-protein substances that help enzymes

Which amino acids can act as acid or base catalysts?

Asp, Glu, His, Lys, Cys, and Tyr

What are coenzymes?

organic cofactors

What are cosubstrates?

loosely bound coenzymes (NAD+)

What are prosthetic groups?

tightly bound coenzymes (FAD)

What are metal ions?

metal cofactors

Fe, Zn, Cu are prosthetic groups
K, Mg, Na
loosely bound

Kd vs Km

Kd:
Binding rate

Km:
Rate of reaction (indirectly measures affinity)
\-- velocity concentration graph --

Main methods of enzyme regulation

- competitive inhibition
- allostery
reversible covalent modification (add phosphate)

Changes to Km with an inhibitor

better the inhibitor the greater the apparent Km
\-- transition state analogue inhibitor has greater Km than substrate analogue inhibitor --

On a enzyme activity-substrate concentration graph what happens when an activator or inhibitor is added

note: activator still sigmoidal

What is required for a kinase catalysis transfer?

ATP, protein-OH

What is required for a phosphatase catalysis?

water, protein-OH

What changes in a protein after phosphorylation?

- size increase
- increased polarity (addition of 2 neg charges)
- ability to make new H bonds

Phsophroylation changes the 3D shape of the enzyme by modifying the catalytic site

A double bond is assumed to be \_______ unless otherwise noted

cis

How to name fatty acids

RULES:
- first carbon is part of carbonyl
- alpha carbon is first in chain (next after carbonyl)
- beta is third carbon
- omega is last carbon
- if there is a trans double bond it must be noted

NAMING:
12:0
- first number \= number of carbons
- second number \= number of double bonds
--\> if there are double bonds a superscript of the carbon that starts the bond is noted (as well as if it is trans)

fatty acid formula

CH3(CH2)nCOO-

What makes increases the strength of fatty acids, and why?

- increased tail length
- saturation

increased van der Waals interactions between tails

Are tri*acyl*glycerols amphipathic?

NO, head groups are not polar enough to be considered amphipathic

Different membrane lipids and their head group differences

*Glycerophospholipids*:
- relatively proportional head group

*Sphingolipids*:
- large head groups made of sugars

*Cholesterol*:
- small head group compared to tail
-weakly amphipathic

Cholesterol is *NEVER* found in \___________

plants or bacteria

How can you transport cholesterol?

make it completely hydrophobic by adding a fatty acid chain to the OH

How to maintain membrane fluidity

in increased temperature:
- add more saturated fatty acids
- add longer fatty acid chains

in decreased temperatures:
- add more unsaturated fatty acids
- add shorter fatty acid chains

What is a porin? What is special about the center of the pore?

Porins are ion channels that enable passive transport, and the center of the three porins are a non-specific water-filled pore.

Different types of active transports

*1°*:
- use ATP as energy source
- can use oxidation

*2°*:
- uses ion gradient as energy source

Epithelial cell transporters

2° symporter on apical side, 1° antiporter on basolateral side

How does NAD go to it's high energy form?

--\> NADH
Accepts two electrons via a hydride ion

net gain:
1 H
2 e-

How does FAD go to it's high energy form?

--\> FADH2
Accepts 2 hydrogen atoms

net gain:
2 H
2 e-

What is catabolism?

the breakdown of complex molecules releasing energy
\-- oxidation

cofactors are reduced

What is anabolism?

The buildup of larger molecules from smaller ones; uses energy
\-- reduction

cofactors are oxidized

Carbohydrates are stored as glycogen in the \________ and \_______________

liver, skeletal muscles

Biochemical standard state ΔG°*'* conditions

- *pH \= 7*
- substrate and product \= 1M
- 25°C
- 1 atm
- (55M water)

What happens to a reaction if ∆G is negative or positive

-∆G \= spontaneous reaction in the forward direction

+∆G \= spontaneous in the reverse direction

Keq and ΔG°' are measures of a reactions tendency to \____________

proceed spontaneously

\-- they are constants --

What is synchronous regulation?

regulating enzymes at the start of the pathway in a similar fashion to those at the end of the pathway, so that they 'turn on and off' at the same time.

3 types of high energy intermediates

1. Electron carriers:
NADH, NADPH, FADH2, FMNH2

2. Nucleotide triphosphates:
NTPs

3. Thioesters

3 main uses of ATP

1. Driving unfavorable reactions (coupling)

2. Movement

3. Active transport

How can coupling drive reactions?

two rxns must be *linked* (share an intermediate) and one reaction must have a sufficiently -ΔG (greater |value| than the other rxn)

Types of regulation inhibition

Product inhibition:
enzyme is inhibited by the products of the reaction pathway (A --\> *B*)

Feedback inhibition:
enzyme is inhibited by a metabolite down the pathway (A --\> B

Feed-forward activation

An enzyme can be activated by a metabolite *upstream*

\-- ensures that intermediates do not build-up --

Reciprocal regulation

Opposing pathways are regulated in inverse directions. Stimulating one pathway inhibits the other

neither pathway can operate at the same time

Where does the energy to make ATP come from in oxidative phosphorylation

free energy from oxidation is used to phosphorylate ADP

Hydrogen concentration across mitochondria

Low in Matrix
High in Intermembrane space

Coenzyme Q is \______-soluble

lipd

Overall electron transport in ETC

*2e-* (--\> NADH/FADH2 --\>) Complex I/II --\> Q --\> Complex III --\> *1e-* Cyt C --\> Complex IV

iron-sulfur clusters

prosthetic group present in complex I, II, and III of electron transport that can carry one electron

-coordinating by cysteines in the protein
-containing equal number of iron and sulfur atoms

What are cytochromes?

hemoproteins (have a heme prosthetic group) and carry 1 electron

Which prosthetic groups in the ETC can transport more than 1 e-?

FMN or FAD

What makes FMN and Q a useful electron carrier compared to other carriers?

It can carry 2 e- and can be partially reduced to accept/release 1 e- at a time

\-- more flexible --

Which complex is FMN found in

Complex I

How many H are generated per complex in ETC?

Complex I: 4
Complex III: 4
Complex IV: 2

What is the benefit of FMN accepting 2 e- from NADH while only releasing 1 e- to FeS?

Prevents e- build up in mitochondria

If you can't \_________ \_________ than you can't move e-

pump protons

How many hydrogens are needed for every ATP synthesized by ATP synthase?

4

How does ATP synthase work?

H+ binds to *F0* causing it to rotate further causing a conformational change in the *F1* catalytic component

\-- energy from conformational change powers ADP --\> ATP

Explain the transfer of e- starting in Complex II

Succinate is oxidized to Fumerate --\> 2 e\- --\> FAD --\> FADH2 --\> Q reduces FADH2

\-- FAD is a prosthetic while succinate is a coenzyme

How is newly sythesised ATP exported (and replenished)?

newly formed ATP in the matrix is exported to the cytosol via the *adenine nucleotide translocase* (*antiporter* of ADP/ATP)

the Pi-H+ *symporter* bring Pi and H+ from intermembrane to matrix

How are oxidative phosphorylation and ATP synthase coupled?

oxidative phosphorylation creates the H+ gradient, and ATP synthase uses the H+ gradient

\-- rate of ATP synthase is dependent on the *magnitude* of the H+ gradient

What is the P/O ratio?

the ratio of oxygen consumed from the ETC to the amount of ATP produced by ATP synthase

What is NADH's P/O ratio?

- 10 H+ gradient produced
- 4H+/ATP therefore

*~2.5*

What is FADH2's P/O ratio?

- 6 H+ gradient
- 4H+/ATP therefore

*~1.5*

How would you reduce the H+ gradient without producing ATP?

Uncoupled systems allow H+ transport across the membrane without the production of ATP

useful when ATP is not being used in cytosol and H+ gradient build-sup

What is produced in uncoupled systems?

H+ ions used to form waster with O2 from ETC

*Heat is generated*!!

What type of tissue are many uncoupled mitochondria systems found

Brown fat

\-- brown because there are many mitochondria compared to white fat, and ETC in mitochondria is rich in iron

Aldohexose is...

glucose

Where does glycolysis occur?

cytosol

What is special about the ATP produced through glycolysis?

*Few* ATP are produced *without* the consumption of O2

Under which conditions does glycolysis occur?

can occur aerobically or anaerobically

\-- typically aerobically --

Type of reactions in first 5 steps of glycolysis (energy investment steps)

1. Phosphorylation (ATP investment)
2. Isomerization
3. Phosphorylation (ATP investment)
4. Lysis
5. Isomerization

Enxymes used to regulate reactions 1-5 in glycolysis

1. Hexo*kinase*
2. isomerase
3. Phosphofructo*kinase*-1 (PFK-1)
4. Aldolase
5. isomerase

What does MgATP do

ATP used in glycolysis, Mg slightly neutralizes ATP making Nu attack from glucose easier

What is a phosphoryl transfer?

add a phosphate using a kinase to generate a phosphate

How does glucose-6-phosphate convert to fructose-6-phosphate?

G6P --\> *open ring* --\> aldehyde --\> *isomerize* --\> keytone --\> *close ring* --\> F6P

Which step is the committed step?

step 3, F6P --\> F-1,6-BP

determines speed of glycolysis

What are the products of the lysis of F-1,6-P?

DHAP (keytone), GAP (aldehyde)

Types of reactions in energy payout steps (step 6-10)

6. Oxidation + Phosphorylation
7. SLP
8. Isomerization
9. Dehydrogenation
10. SLP

Enzymes used to regulate reactions 6-10 in glycolysis?

6. GAP dehydrogenase (GAPDH)
7. Phosphoglycerate *kinase*
8. \---- [mutase]
9. \---- [enolase]
10. Pyruvate kinase

Which steps are energy capture steps?

6 \-- NADH produced
7 \-- ATP produced
10 \-- ATP produced

How is the first ATP gained in glycolysis?

made through the breaking of acyl phosphate in step 7

Why is the creation of PEP in step 9 from 2-Phosphoglycerate not an energy capture step?

PEP is not released; it is used up in the next step

Why is regulation important?

- ensure cells needs are met
- fuel wasted
- there are appropriate levels of intermediates for other purposes

Glucose-6-phosphate is regulated via \____________ inhibition

product

PFK-1 is regulated via \____________ inhibition

allosteric

\-- ADP/AMP and PEP --
PEP inhibits
ADP/AMP activates

Pyruvate kinase is regulated via \___________ inhibition

allosteric
(part of reciprocal regulation - glycolysis vs gluconeogenesis)

\-- ATP and F-1,6-BP --
ATP inhibits
F-1,6-BP activates

production of lactate

Pyruvate + NADH + H --(lactate dehydrogenase)--\> L-Lactate

Why is lactate considered a "dead-end" product in anaerobic acitivty?

It is produced by a cell but cannot be used by cell

What is the purpose of the Plasmamembrane symporter?

transport lactate and H+ out of muscles and into blood

lower blood pH \= easier oxygen drop off in tissues