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Bio98 Quiz 1 Review

Rough Schedule:
Useful techniques for Critical Thinking questions (10 mins)
Energetics of reactions(30 mins)
Enzymes and Reaction Rates (25 mins)
pH of Amino acids, Functional Groups (30 mins)
3D Structure and Function of Proteins (25mins)
*Evaluations will be passed out towards the end of the review.
Questions? Please ask along the way!
*Pictures and concepts in the following presentation have been cited from the Lehninger textbook and
-Biochemistry-Lectures Copyright Pavan Kadandale and Lehninger et al- All rights reserved.

Francesco
Nguyen

Preparing to Think Critically

1.
2.
3.
4.
5.
6.

Actively read and understand concepts


Ask Questions
Research, discuss (yourself + others)
Applications
Practice Questions
Have fun!

Approaching Critical Thinking Questions


1.
2.
3.
4.

Differentiate between givens and unknowns


a.
Whats significant?
Draw or write to relate concepts to the problem
Justify your conclusion with those concepts
Smile

Example Problem: Energetics of Reactions


Under standard conditions, ATP hydrolysis (G) is
-30.5 kJ/mol. Use the resulting plot below to
explain why metabolism is regulated to keep the
ratio of ATP/ADP high in the cell.
AT
P

0.2

AD
P

0.2

2.2

4.2

5.0

25

Using Our Techniques

Under standard conditions, ATP hydrolysis (G)


is -30.5 kJ/mol. Use the resulting plot below to
explain why metabolism is regulated to keep the
ratio of ATP/ADP high in the cell.
ATP IS IMPORTANT: coupled with countless
reactions in the cell!

Energetics of reactions
Before we answer that question, lets do a little review.

Delta G: Free Energy Change

Delta G (G):
energy change given in
varying conditions (cell =
dynamic)
function of temperature,
concentrations
Represented by:
G=G+RTln[(prod/react)]
Q= [Product]i/[Reactant]i
Direction the reaction will go
given all conditions

Standard Delta G (G):


tells us nature of products and
reactants
direction reactants want to go
at standard conditions
Represented by:
G= -RTln(Keq)
Ratio of products and reactants AT
EQUILIBRIUM

Demonstration:

ATP-->ADP + Pi

VS. (MORE) ATP ADP+Pi

Concept 1: one way to increase magnitude (-)G is


increase intracellular reactant concentrations.
APPLICATION = Glucose (blood)

Back to Our Problem:


Under standard conditions, ATP
hydrolysis (G) is -30.5 kJ/mol. Use the
resulting plot below to explain why
metabolism is regulated to keep the ratio
of ATP/ADP high in the cell.

ATP ADP+ Pi
ATP
(m
M)

0.2

AD
P

0.2

2.2

4.2

5.0

25

Answer?
The cell wants to keep ATP at a
high concentration so that
because ATP hydrolysis is lower
when there are more products
than reactants. Thus, energy
available to the cell is higher
when ATP/ADP ratio is greater.
G=G+RTln[(prod/react)]
Given: standard delta G <0
RTln(<<1) <0 -->More reactants
RTln (>>1) >0 less reactants,
less potential energy (delta G is
smaller)

Recap

Understand context of a cell versus


standard conditions
Be able to think about multiple
scenarios

QUESTIONS?

One more Delta G

If the reaction A+B C has a G= -25 kJ/mol. Which


molecules will be at the highest concentrations at
equilibrium?
A. Molecule A
B. Molecule B
C. A and B
D. Molecule C
E. Cannot be determined. We dont know other conditions

Questions?

Last G Problem
Consider the hypothetical transformation of
X Y, for which G= 10.0 kJ/mol
a. what is the ratio of [Y]/[X] at equilibrium?
b. If you add a molecule Z, that reacts with Y, what will happen to the G of X
Y?
c.

The reaction Y+Z A is catalyzed by enzyme YA in order to see the reaction


in a timely fashion (seconds rather than days). If you add an inhibitor to block
the function of YA, what will happen to your products in that time?

Answer 1:

Consider the hypothetical transformation of


X Y, for which G= 10.0 kJ/mol
a. what is the ratio of [Y]/[X] at equilibrium?
. Ans: X!
a. If you add a molecule Z, that reacts with Y, what will happen to the G of X
Y?
b. The reaction Y+Z A is catalyzed by enzyme YA in order to see the reaction
in a timely fashion (seconds rather than days). If you add an inhibitor to block
the function of YA, what will happen to your products in that time?

Answer 2

Consider the hypothetical transformation of


X Y, for which G= 10.0 kJ/mol
a. what is the ratio of [Y]/[X] at equilibrium?
. Ans: X!
a. If you add a molecule Z, that reacts with Y, what will happen to the G of X
Y?
. Ans: G will become more negative, goes forward
a. The reaction Y+Z A is catalyzed by enzyme YA in order to see the reaction
in a timely fashion (seconds rather than days). If you add an inhibitor to block
the function of YA, what will happen to your products in that time?

Answer 3

Consider the hypothetical transformation of


X Y, for which G= 10.0 kJ/mol
a. what is the ratio of [Y]/[X] at equilibrium?
. Ans: X!
a. If you add a molecule Z, that reacts with Y, what will happen to the G of X
Y?
. Ans: G will become more negative, goes forward
a. The reaction Y+Z A is catalyzed by enzyme YA in order to see the reaction
in a timely fashion (seconds rather than days). If you add an inhibitor to block
the function of YA, what will happen to your products in that time?
. Ans: it will take a while before you see any product, but once at equilibrium,
ratio will be the same!

Energetics: Coupling Reactions

Is it feasible to only increase concentrations in cells?


No!
Concept 2: Thats why we couple delta G!
coupling transport (membranes)
gradient formation
glucose transport in intestine
Biochem reactions, and more

Energetics: Coupling Reactions

Is it feasible to only increase concentrations in cells?


No!
Concept 2: Thats why we couple delta G!
coupling transport (membranes)
gradient formation
glucose transport in intestine
Biochem reactions, and more

Your favorite reaction (so far)

1) Glucose + Pi glucose-6-phosphate +H20


G= 13.8 kJ/mol
2) ATP +H20 ADP+Pi
G= -30.5 kJ/mol
3) Sum: Glucose +ATP glucose-6-phosphate +ADP
G sum = -16.2 kJ/mol (thermodynamically favored forward)

Enzymes

Do enzymes shift the equilibrium?


o

No, enzymes do NOT shift the equilibrium

So what can they do?


o

All they can do is SPEED UP the reaction

How do they do that?


o
o

by LOWERING the Ea (activation energy) orient molecules in a way that makes them
easier to react
stabilizes T.S.

They are SPECIFIC!


o

What accounts for the SPECIFICITY of an enzyme?

its functional group diff. interactions

Enzyme Active Sites

What determines the enzymes interaction with its


substrate?
o
o

Am. Acid residues of the active site (functional groups)


Enzyme shape (defined 3D structure)

Active site residues initiate rxns and stabilize TS


CHIRAL and sensitive to chirality

NOTE!

Enzymes CANNOT change whether a reaction is favorable or not.

All an enzyme will do is:


o
make the reaction more kinetically favorable
o
by lowering the Ea of the rxn

ex. if a reaction doesnt proceed because its not favorable ( G

=+)
o adding an ezyme will not make the reaction go

Enzyme Problem

Suppose an enzyme undergoes a mutation that no longer


allows it to form a conformation change. How will this affect
a reaction?
Will the rxn occur?
o

no, a conformational change is needed for the substrate to bind to the enzyme (perfect,
specific fit)

What would happen to the rate of the reaction?


o

undergoes normal rate (wont speed up, bc no enzyme-substrate complex forms)

no Ea lowered

Enzyme Problem 2!

Lets say an active site is made up of amino acid residues


that are all in a line. However, these residues now become
spaced out throughout the enzyme (could be due to a
mutation).
How will this affect the enzymes ability to bind the
substrate?
o it wont be able to bind to its normal substrate
What does it have to do in order to bind?
o
o

can bind with a chaperone protein


Chaperone proteins stabilize enzymes so it wont mutate/denature

Enzyme Problem 3!

For the conversion of glucose to G6P, hexokinase is used.


If more hexokinase was injected into the body, how would
this change the delta G of the reaction?
The delta G is not affected because it is independent
from [enzyme]

Enzyme Problem 4 (last one)

To speed up the rate of a glucose rxn, enzyme WAT is


used. To speed up rate of galactose reaction, enzyme SUP
is used. How would you speed up the rate of a lactose (glc
+ galac) rxn?
adding WAT/SUP wont work
you need to find a SPECIFIC enzyme for lactose
Looking at lecture, why cant our body digest L-Glc but it
can digest D-Glc?
Hexokinase SPECIFIC to D-GLucose! active sites
are CHIRAL and sensitive to chirality

Amino Acids, and Functional Groups


1.
2.
3.

Amino acid groups (sticky note)


pH/Titration curves
Calculating overall charge

Why are pH curves important?


Maybe a few reasons:
1.
Structure = function (extremely critical) any change in pH or temperature leads to _____
2.
Knowing pH isoelectric points tells us:
a.
Types of interactions
b.
Location in protein/cell
c.
biochemical modeling

Modeling of Proteins

location?
sequence?
bonds?
o
interactions

charges

Common Questions
1.

Should we know the name and structures of all the amino acids?

At least know: groups they are categorized in (hydrophobic, polar, charged)

Amino Acid Groups: Sample Question

Name three amino acids that could


be located in the hydrophobic
region of a transmembrane
protein?
What about the amino acids in the
inside of a ionic channel in
neurons?
Answer:

Sample Question 2
What is the isoelectric point of cysteine?

K2

Protein/pH Question

You and a group of friends in your research lab decide to


purify a secretion protein that is important in innate immune
defense. This class of protein normally has positively
charged amino acids such as histidine and arginine. Thus,
you decide to purify it using a negatively charged resin in
the hopes to eventually figure out the entire sequence. At
this point, the buffers were added with a pH of 10 and a salt
concentration of 300 mM NaCl and the lysed solution was
run through the resin. VERY little protein was collected.
Why?

Whats a better way to purify the protein from the bacterial cell?

Questions?

After successfully purifying your defensive protein, you and


your lab partners hypothesize where the protein is secreted
and used in the body.
Ideas?
Mucous membranes of the intestine
Saliva
Skin
Respiratory mucous membranes

What is the overall charge of a protein that has the following sequence at pH=7?
Glu-Gly-Ala-Lys
Which charge resin could you purify this with?
Positive, Negative, you cant?

3D Structure and Function of


Proteins

Anfinsens Experiment

What did he want?


o

Wanted to unfold a protein

So what did he do?


o

he heated the protein

denatured H bonds/weak bonds

What happened?
o

Proteins lost activity

How a protein folds


is determined by
PRIMARY
STRUCTURE!!

He left it to cool over time. What happened?


o

Protein refolded (deduced that bc protein activity came back over time)

Jeoffrey
(Activity)

Cersei
(Activity)

Nothing

400

380

Heat

22

26

Heat +
Recovery

379

29

Heat +
Recovery
+ Jamie

379

368

Heat +
Jamie

26

28

Heat +
Jamie +
Recover

378

30

Given the following


hypothetical enzymes
and observations, what
could be said about the
enzymes, Jeoffrey,
Cersei, and Jamie?
Jeoffery is capable of refolding
himself. Cersei cant. Cersei
requires the chaperone protein,
Jamie in order to refold. Jamie
cannot fold by itself.

Primary Structure

Protein folding information contained in the primary am.


acid seq.
Made up of peptide bonds (planar)
o

can rotate due to resonance

Secondary Structures (3D)

A - helix
o
formation of H bonds stabilizes struc. by orienting hydrophobic regions outwards
o
this stabilizes the struc bc if outward, they would have constant replusion with water

gives them a mobile struc and its struc is specific to protein function
o
use of internal H-bonds (between C & N terminus backbone) causes a-helix to form helix
struc
o
R- groups parallel

Secondary Structures

B- pleated sheet
o
allows the structure to function fully in a zig zag and alternating sequence

H-bonds can interact with each other laterally in an anti confomation


o
parallel/anti-parallel

Tertiary Structure

Hydrophobic interactions (H bonds)/weak bonds


determines the folding of tertiary structures
o

driving force for folding

combines A & B structures

Problem!

If a cell were going to create a protein that exists within the


plasma membrane, how would you expect the protein to
fold?
It would fold so that its hydrophobic amino acids are
facing outwards so that it is able to interact with the
nonpolar fatty acid tails that make up the interior of the
plasma membrane

And thats it!


QUESTIONS!?!? Ask now :)
Extra Resources:
Office hours on Tuesday, day before your exam.
Check: https://sites.google.com/a/uci.edu/biotutor/

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